High-molecular-weight fucans for treating fibrous adhesions and other diseases and conditions

ABSTRACT

High-molecular-weight fucan compositions comprising a therapeutically effective, medically acceptable fucan in a composition comprising wherein the fucan, for example, has a molecular weight distribution in which more than 60% w/w of the composition has a molecular weight above 100 kDa.

CLAIM FOR PRIORITY

The present application claims the benefit of United States provisionalpatent application Ser. No. 62,711,364, filed Jul. 27, 2018; UnitedStates provisional patent application Ser. No. 62,711,372, filed Jul.27, 2018; U.S. provisional patent application No. 62/711,335, filed Jul.27, 2018; U.S. Provisional Patent Application Ser. No. 62/713,399, filedAug. 1, 2018; U.S. provisional patent application No. 62/722,135, filedAug. 23, 2018; U.S. provisional patent application No. 62/755,311, filedNov. 2, 2018; U.S. provisional patent application No. 62/793,514, filedon Jan. 17, 2019; U.S. provisional patent application No. 62/861,223,filed Jun. 13, 2019; U.S. Provisional Patent Application Ser. No.62/713,392, filed Aug. 1, 2018; U.S. provisional patent application No.62/713,413, filed Aug. 1, 2018; U.S. provisional patent application No.62/722,137, filed Aug. 23, 2018; U.S. provisional patent application No.62/755,318, filed on Nov. 2, 2018; U.S. provisional patent applicationNo. 62/861,228, filed Jun. 13, 2019; U.S. Provisional Patent ApplicationSer. No. 62/755,328, filed Nov. 2, 2018; U.S. provisional patentapplication No. 62/793,654, filed Jan. 17, 2019; and, U.S. provisionalpatent application No. 62/861,235, filed Jun. 13, 2019, all of whichapplications are incorporated herein by reference in their entirety.

BACKGROUND

Fucans (including fucoidan) are sulfated polysaccharides. In generalterms, this means that they are molecules made up of a number of sugargroups, and also have sulfur atoms attached to the sugar groups. Themain sugar group is called “fucose”, which is sugar that has 6 carbonatoms and has the chemical formula C₆H₁₂O₅. “Fucoidan” (or fucoidin)indicates fucans derived from brown algae (seaweed). Fucans can existalone, or in a mixture of other sugars, for example in a mixture ofsugars such as xylose, galactose, glucose, glucuronic acid and/ormannose. These other sugars may be extracted from the seaweed or othersource with the fucan. Although fucans are currently derived fromnatural sources such as the brown algae (seaweeds), sea cucumbers, etc.,mentioned herein, “fucan” includes polymer molecules having the chemicaland structural motifs of the fucans as discussed herein regardless ofthe ultimate source(s) of the fucans.

Fucoidan can be obtained from a variety of species of brown algaeincluding but not limited to: Adenocystis utricularis, Ascophyllumnodosum, Chorda filum, Cystoseirabies marina, Durvillaea antarctica,Ecklonia kurome, Ecklonia maxima, Eisenia bicyclis, Fucus evanescens,Fucus vesiculosis, Hizikia fusiforme, Himanthalia Elongata,Kjellmaniella crassifolia, Laminaria brasiliensis, Laminariacichorioides, Laminaria hyperborea, Laminaria japonica, Laminariasaccharina, Lessonia trabeculata, Macrocystis pyrifera, Pelvetiafastigiata, Pelvetia Canaliculata, Saccharina japonica, Saccharinalatissima, Sargassum stenophylum, Sargassum thunbergii, Sargassumconfusum, Sargassum fusiforme and Undaria pinnatifida. These exemplaryspecies are all from the taxonomic class Phaeophyceae and the majorityof these species fall into the families of Fucales and Laminariaceae.

Fucans including fucoidan have been shown to be efficacious in servingto inhibit, prevent, remove, reduce, or otherwise treat the formation offibrous adhesions. They have also found use in the treatment of otherrelated diseases and conditions.

Thus, there has gone unmet a need for compositions comprising fucanshaving desired high-molecular-weights, including in some embodimentssuch compositions being modified to have desired sulfation levels and/ormedically viable, low endotoxin levels. The present compositions,systems and methods, etc., provide these and/or other advantages.

SUMMARY

The present compositions, systems, devices, materials and methods, etc.,provide high-molecular-weight fucans. Such high-molecular-weight fucanscan be obtained from feedstock fucan compositions or other starting orinitial fucan compositions that have fucans with a broad molecularweight distribution comprising a desired high-molecular-weightsegment/portion (i.e., broad molecular weight fucan compositions fromwhich the high-molecular weight fucans can be derived; such startingfucan compositions may or may not be crude or have been previouslyprocessed or purified). The desired high-molecular-weight fucan has amolecular weight distribution consisting essentially of the desiredhigh-molecular-weight segment/portion of the starting fucan broadmolecular weight distribution wherein a substantial quantity of thebroad molecular weight distribution at the low molecular weight end hasbeen eliminated, suppressed, or otherwise attenuated such that anyremaining amounts are inconsequential.

In some aspects, the compositions, systems, methods, etc., hereincomprise high-molecular-weight fucans such as fucoidans can comprise,consist essentially of, or consist of. a molecular weight distributionwherein at least 60% w/w of the distribution can be greater than 100 kDawhen measured using an aqueous gel permeation chromatography set upconsisting essentially of:

-   -   one 300 mm analytical gel permeation chromatography column with        a 7.8 mm inner diameter packed with hydroxylated        polymethacrylate-based gel, having an effective molecular weight        range of can be between about 50 kDa and about 5,000 kDa, one        300 mm analytical gel permeation chromatography column with a        7.8 mm inner diameter packed with hydroxylated        polymethacrylate-based gel, having an effective molecular weight        range of can be between about 1 kDa and about 6,000 kDa and one        40 mm guard column with a 6 mm inner diameter packed with        hydroxylated polymethacrylate-based gel, the two analytical gel        permeation chromatography columns and the one guard column        contained in a column compartment at about 30° C.;    -   a refractive index detector at about 30° C.;    -   0.1M sodium nitrate mobile phase run at 0.6 mL/min; and    -   quantification against a peak molecular weight standard curve        consisting essentially of a first dextran standard with a peak        molecular weight of about 2,200 kDa, a second dextran standard        with a peak molecular weight of can be between about 720 kDa and        about 760 kDa, a third dextran standard with a peak molecular        weight can be between about 470 kDa and about 510 kDa, a fourth        dextran standard with a peak molecular weight can be between        about 370 kDa and about 410 kDa, a fifth dextran standard with a        peak molecular weight can be between about 180 kDa and about 220        kDa, and a sixth dextran standard with a peak molecular weight        can be between about 40 kDa and 55 kDa.

In some embodiments, at least about 70% w/w, 80% w/w, 90% w/w, 93% w/w,94% w/w, 95% w/w, 97% w/w, 98% w/w, or 99% w/w of the distribution canbe greater than 100 kDa. The weight average molecular weight can bebetween about 100 kDa and 10,000 kDa; between about 140 kDa and 8,100kDa; between about 370 kDa and 8100 kDa; between about 370 kDa and 5300kDa; between about 370 kDa and 8100 kDa; between about 370 kDa and 5300kDa; between about 370 kDa and 1900 kDa; between about 590 kDa and 1600kDa; between about 590 kDa and 1600 kDa; or between about 860 kDa and1600 kDa. In some embodiments, the weight average molecular weight canbe about 1,100 kDa, about 1,200 kDa, or about 1,300 kDa. The numberaverage molecular weight can be between about 50 kDa and 3,000 kDa;between about 60 kDa and 2,000 kDa; between about 140 kDa and 2,000 kDa;between about 140 kDa and 520 kDa; or between about 230 kDa and 450 kDa.At least 55% w/w, 71% w/w, or 91% w/w of the distribution can be greaterthan about 200 kDa. At least 22%, 54% w/w, or 90% w/w of thedistribution can be greater than about 500 kDa.

In some embodiments, the high-molecular-weight fucans can consistessentially of, comprise, or consist of, a molecular weight distributionwherein can be between about 61% w/w and 80% w/w of the distribution canbe between about 200 kDa and 1600 kDa when measured using an aqueous gelpermeation chromatography set up as set forth above and elsewhereherein. The high-molecular-weight fucans can consist essentially of,comprise, or consist of, a molecular weight distribution wherein atleast 60% w/w of the distribution can be greater than about 1600 kDawhen measured using an aqueous gel permeation chromatography set up asset forth above and elsewhere herein.

The sulfate content can be between about 20% w/w and 60% w/w, about 30%w/w and 55% w/w, or about 32% w/w and 52% w/w. The total carbohydratecontent can be between about 27% w/w and 80% w/w. The total fucosecontent as a percentage of the total carbohydrate content can be atleast about 30% w/w, 50% w/w, 70% w/w, 80% w/w, 90% w/w or 95% w/w. Thetotal galactose content as a percentage of the total carbohydratecontent can be below about 60% w/w, or can be between about 2% w/w and20% w/w, or can be below about 10% w/w. The total of glucuronic acid,mannose, rhamnose, glucose and xylose content as a percentage of thetotal carbohydrate content can be below about 30% w/w.

The high-molecular-weight fucans when dissolved in water at aconcentration of 50 mg/mL has a viscosity of can be between about 4 cPand 50 cP; between about 10 cP and 40 cP; or between about 15 cP and 30cP. The high-molecular-weight fucans can be a white solid, and whendissolved in water at a concentration from 1 mg/mL through 100 mg/mLforms a solution that can be one of clear-colorless. The fucan cancomprise less than about 5% w/w or 2% w/w acetyl content. The fucan cancomprise an acetyl content of substantially 0% w/w when measured by 2D¹H-¹³C heteronuclear multiple quantum coherence at 70° C. with solventsignal suppression on a 600 MHz spectrometer equipped with 5-mm coldprobe, in the range from 10-30 ppm in the carbon dimension, in 8increments of 256-512 scans each.

Also included herein are methods, including methods that can comprisemaking or using the high-molecular-weight fucans herein, including fortreating fibrous adhesions. Further included herein are medicallyacceptable fucan compositions that can comprise a therapeuticallyeffective amount of the high-molecular-weight fucans in a medicallyacceptable buffer or diluent. Methods also include treating a conditionor disease in an animal that can comprise selecting the medicallyacceptable fucan compositions herein to treat the condition or diseaseand administering a therapeutically effective amount comprising betweenabout 0.5 mg/kg and 50 mg/kg; 0.04 mg/kg and 25 mg/kg; 0.2 mg/kg and 10mg/kg; 1 mg/kg and 5 mg/kg; 1.5 mg/kg and 3 mg/kg; 5 mg/kg and 10 mg/kg.

The condition or disease can be a fibrous adhesion at a target site inthe animal, and the administering can comprise administering thetherapeutically effective amount to the target site.

The medical compositions can be between about 0.02 mg/mL and 100 mg/mLof the high-molecular-weight fucans, wherein the medical compositions isconfigured and composed to treat a disease or condition in an animal.The medical compositions can also be between about 0.5 mg/mL and 5mg/mL, or about 2.5 mg/mL, of the high-molecular-weight fucans.

The medical compositions can be a medical device including a liquidmedical device. The medical compositions can be pharmaceuticalcompositions, which can be liquid pharmaceutical compositions.

The methods herein also include use of a dosage range comprising betweenabout 0.01 mL/kg and 15 mL/kg; about 0.03 mL/kg and 4 mL/kg; about 0.06mL/kg and 2 mL/kg; or, about 2 mL/kg and 4 mL/kg of the medicalcompositions to treat a disease or condition in an animal.

The methods for treating fibrous adhesions in a patient can compriseadministering the medical compositions to a target site in the patient.The target site can be a surgical site and the administering can beperformed at least one of a) after opening a surgical wound at thesurgical site, b) during surgery, and c) after closing the surgicalwound. The administering can be performed after surgery and beforeclosing the surgical wound. The administering can take less than 3minutes, 2 minutes or 1 minute. The target site can be at least one of alesion, abrasion and injury site. The target site can be at least one ofa pelvic cavity, an abdominal cavity, a dorsal cavity, a cranial cavity,a spinal cavity, a ventral cavity, a thoracic cavity, a pleural cavity,a pericardial cavity, skin, a joint, a muscle, a tendon and a ligament.

In further embodiments, the methods herein include methods for obtaininga high-molecular-weight fucans. Such methods can comprise:

-   -   providing in a starting solution a starting fucan compositions        having a broad molecular weight distribution comprising a        desired high-molecular-weight fucans segment;    -   subjecting the starting solution to a first tangential flow        filtration across a first higher molecular weight cutoff        tangential flow filtration filter to produce a first permeate        fucan compositions; and    -   subjecting the first permeate fucan compositions to a second        tangential flow filtration across a second lower molecular        weight cutoff tangential flow filtration filter to produce a        second retentate fucan compositions consisting essentially of        the desired high-molecular-weight fucans.

The methods further can comprise collecting the second retentate fucancompositions consisting essentially of the desired high-molecular-weightfucans, and the first higher molecular weight cutoff tangential flowfiltration filter has a higher molecular weight cutoff of can be betweenabout 50 kDa and about 1000 kDa and the second lower molecular weightcutoff tangential flow filtration filter has a lower molecular weightcutoff of can be between about 30 kDa and about 100 kDa. The highermolecular weight cutoff can be about 300 kDa and the lower molecularweight cutoff can be about 100 kDa.

Methods for obtaining a high-molecular-weight fucans can comprise:

-   -   providing a starting fucan compositions having a broad molecular        weight distribution comprising a desired high-molecular-weight        fucans segment in a starting solution;    -   subjecting the starting solution to tangential flow filtration        across a first lower molecular weight cutoff tangential flow        filtration filter to produce a first retentate fucan        compositions; and    -   subjecting the first retentate fucan compositions to tangential        flow filtration across a second higher molecular weight cutoff        tangential flow filtration filter to produce a second permeate        fucan compositions consisting essentially of the desired        high-molecular-weight fucans.

The methods further can comprise collecting the second permeate fucancompositions consisting essentially of the desired high-molecular-weightfucans. The first tangential flow filtration can comprise diafilteringthe starting solution across the first lower molecular weight cutofftangential flow filtration filter. The second tangential flow filtrationcan comprise diafiltering the first retentate fucan compositions acrossthe second higher molecular weight cutoff tangential flow filtrationfilter. The first lower molecular weight cutoff tangential flowfiltration filter has a lower molecular weight cutoff of can be betweenabout 30 kDa and about 100 kDa and the second higher molecular weightcutoff tangential flow filtration filter has a higher molecular weightcutoff of can be between about 50 kDa and about 1000 kDa. The lowermolecular weight cutoff can be about 100 kDa and the higher molecularweight cutoff can be about 300 kDa.

Methods for obtaining a high-molecular-weight fucans can comprise:

-   -   providing a starting fucan compositions having a broad molecular        weight distribution comprising a desired high-molecular-weight        fucans segment in a starting solution, the starting fucan        compositions can comprise low atomic weight cations ionically        bound to the sulfate groups on fucan in the compositions; and    -   subjecting the starting solution to tangential flow filtration        against a cationic additive solution can comprise a cationic        additive having a greater molecular weight than the low atomic        weight cations to produce a retentate fucan compositions        consisting essentially of the desired high-molecular-weight        fucans.

The methods further can comprise collecting the retentate fucancompositions consisting essentially of the desired high-molecular-weightfucans. The low atomic weight cations comprise at least one of an alkalimetal, an alkaline earth metal, aluminum and ammonium. The cationicadditive can comprise at least one of choline, polyvinylpyrrolidone,taurine, polyamine, chitosan, histone, and collagen. The methods furthercan comprise adding to the starting solution the cationic additivebefore subjecting the starting solution to tangential flow filtration.The tangential flow filtration can comprise diafiltering the startingsolution against the cationic additive solution. The methods stillfurther can comprise removing the cationic additive by diafiltering theretentate fucan compositions against a salt solution over a secondtangential flow filtration filter having a molecular weight cutoff thatcan be lower than a molecular weight cutoff of the first tangential flowfiltration filter.

The salt solution can comprise a chloride, bromide, iodide, fluoride,sulfate, sulfite, carbonate, bicarbonate, phosphate, nitrate, nitrite,acetate, citrate, silicate and/or cyanide of an alkali metal, alkalineearth metal, aluminum and/or ammonium. The methods can also compriseremoving salt by diafiltering the retentate fucan compositions against alow-ionic strength solution.

Methods for obtaining a high-molecular-weight fucans can comprise:

-   -   providing a centrifuge container can comprise a bottom end and a        top end and a permeable barrier therebetween, the permeable        barrier can comprise a gradient material therebetween;    -   placing a starting fucan compositions having a broad molecular        weight distribution comprising a desired high-molecular-weight        fucans segment in the centrifuge container and above the        permeable barrier; and    -   centrifuging the centrifuge container for a period of time        sufficient to produce a precipitate consisting essentially of        the desired high-molecular-weight fucans.

The methods further can comprise collecting the desiredhigh-molecular-weight fucans from the centrifuge container. Thepermeable barrier can comprise a single segment of gradient material.The permeable barrier can comprise a plurality of segments of gradientmaterial. The gradient material can comprise at least one of sucrose,polysucrose, glycerol, sorbitol, CsCl, Cs₂SO₄, KBr, diatrizoate,Nycodenz® and iodixanol. The centrifugal force can be between about10,000 gravities to about 1,000,000 gravities. The centrifugal force canbe between 60,000 gravities to about 500,000 gravities.

Methods for obtaining a high-molecular-weight fucans can comprise:

-   -   providing a centrifuge container can comprise a bottom end and a        top end;    -   placing a starting fucan compositions in a starting solution,        having a broad molecular weight distribution comprising a        desired high-molecular-weight fucans segment in the centrifuge        container; and    -   centrifuging the centrifuge container for a period of time        sufficient to produce a precipitate consisting essentially of        the desired high-molecular-weight fucans.

The methods further can comprise collecting the desiredhigh-molecular-weight fucans as a precipitate from the centrifugecontainer. The centrifugal force can be between about 60,000 gravitiesto about 1,000,000 gravities. The centrifugal force can be between200,000 gravities to about 500,000 gravities.

Methods for obtaining a high-molecular-weight fucans can comprise:

-   -   subjecting a starting fucan compositions having a broad        molecular weight distribution comprising a desired        high-molecular-weight fucans segment to gel electrophoresis        wherein the starting fucan compositions can be displaced        according to mass-to-charge ratio across an electrophoresis gel;    -   selecting a portion of the electrophoresis gel consisting        essentially of the desired high-molecular-weight fucans; and    -   extracting the desired high-molecular-weight fucans from the        selected portion of the electrophoresis gel.

The subjecting the starting fucan compositions to gel electrophoresiscan comprise applying a potential difference across the electrophoresisgel can be between about 10 Volt/cm and 200 Volt/cm. The electrophoresisgel can comprise at least one of agarose, polyacrylamide,polydimethylacrylamide and starch. The electrophoresis gel further cancomprise at least one of tris-acetate EDTA, tris-borate EDTA andphosphate buffered saline. Extracting the desired high-molecular-weightfucans from the selected portion of the electrophoresis gel can compriseagitating the selected portion of the electrophoresis gel in a solvent.The solvent can comprise at least one of water, methanol, ethanol andisopropanol.

Methods for obtaining a high-molecular-weight fucans can comprise:

-   -   providing a starting fucan compositions having a broad molecular        weight distribution comprising a desired high-molecular-weight        fucans segment, and an ion exchange macroporous resin; and    -   subjecting the starting fucan compositions to ion exchange with        the ion exchange macroporous resin to obtain an ion exchange        treated fucan compositions consisting essentially of the desired        high-molecular-weight fucans.

The methods further can comprise collecting the desiredhigh-molecular-weight fucans from the ion exchange treated fucancompositions. Providing the starting fucan compositions further cancomprise desalting the starting fucan compositions before subjecting thestarting fucan compositions to ion exchange. A mass ratio of thestarting fucan composition:ion exchange macroporous resin can be betweenabout 1:100 and about 10:1. The mass ratio can be between about 1:10 andabout 5:1. The starting fucan compositions can be subjected to ionexchange for a period of can be between about 5 minutes and about 100hours. The ion exchange macroporous resin can comprise at least one ofan anion exchange macroporous resin and a mixed charge macroporousresin. The anion exchange macroporous resin can be a strong basemacroporous resin. The strong base macroporous resin can comprisequaternary amine groups. The anion exchange macroporous resin can be aweak base macroporous resin. The weak base macroporous resin cancomprise at least one of primary, secondary or tertiary amine groups.The ion exchange macroporous resin can comprise at least one of styrene,agarose, dextran, acrylate, methacrylate, methyl methacrylate, butylmethacrylate, divinylbenzene, cellulose, silica, and ceramic. The ionexchange macroporous resin has a pore size of can be between about 5 nmand about 1000 nm, about 10 nm and about 100 nm, or about 15 nm andabout 50 nm. The ion exchange macroporous resin can have an exclusionlimit of can be between about 50 kDa and about 50,000 kDa, about 1,000kDa and about 9,000 kDa, or about 100 kDa and about 1,000 kDa. Thestarting fucan compositions can be subjected to anion-exchange for aperiod of can be between about 5 minutes and about 100 hours or betweenabout 1 hour and about 30 hours.

Methods for obtaining a high-molecular-weight fucans can comprise:

-   -   providing a starting fucan compositions with a broad molecular        weight distribution comprising a desired high-molecular-weight        fucans segment in a starting solution, and a gel media;    -   subjecting the starting solution to preparative gel permeation        chromatography, wherein the starting fucan compositions can be        displaced from a first input end to a second output end across        the gel media according to molecular weight; and    -   collecting from the second output end at least one aliquot        consisting essentially of the desired high-molecular-weight        fucans segment.

The methods further can comprise collecting multiple aliquots andcombining the aliquots. The gel media can be contained in a column. Thegel media can comprise at least one of polyhydroxymethacrylate,sulfonated styrene-divinylbenzene, silica, a hydrophilic bonded phase orpolymer, polystyrene, divinylbenzene, methacrylate, methyl methacrylate,butyl methacrylate, cellulose, ceramic, agarose and dextran. The gelmedia has a pore size of can be between about 3 nm and about 3000 nm, 3nm and about 3000 nm, about 5 nm and about 10,000 nm, about 10 nm andabout 100 nm, about 50 nm and about 500 nm, about 200 nm and about 2,000nm, or about 500 nm and about 5,000 nm. The gel media has an exclusionlimit of can be between about 100 Da and about 100,000 kDa, about 100kDa and about 30,000 kDa, about 1,000 kDa and about 100,000 kDa, about1,000 kDa and about 10,000 kDa, or about 5,000 kDa and about 50,000 kDa.

These and other aspects, features and embodiments are set forth withinthis application, including the following Detailed Description andattached drawings. Unless expressly stated otherwise, all embodiments,aspects, features, etc., can be mixed and matched, combined and permutedin any desired manner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts an exemplary two-filter system for thesegmentation of a starting fucan composition on the basis of molecularweight using sequential tangential flow filtration, the starting fucanhaving a broad molecular weight distribution.

FIG. 2 schematically depicts an exemplary further embodiment of atwo-filter system for the segmentation of a starting fucan compositionon the basis of molecular weight using sequential tangential flowfiltration, the starting fucan having a broad molecular weightdistribution.

FIG. 3 schematically depicts an exemplary system for obtaining a desiredhigh-molecular-weight fucan from a starting fucan composition usingcation-augmented tangential flow filtration, the starting fucan having abroad molecular weight distribution.

FIG. 4 schematically depicts an exemplary system for centrifugallyprecipitating a high-molecular-weight fucan from a starting fucancomposition using a multi-segment barrier of gradient material, thestarting fucan having a broad molecular weight distribution.

FIG. 5 schematically depicts an exemplary system for centrifugallyprecipitating a high-molecular-weight fucan from a starting fucancomposition using a single segment barrier, the starting fucan having abroad molecular weight distribution.

FIG. 6 schematically depicts an exemplary system for obtaining ahigh-molecular-weight fucan from a starting fucan composition by gelelectrophoresis-extraction, the starting fucan having a broad molecularweight distribution.

FIG. 7 schematically depicts an exemplary system for obtaining ahigh-molecular-weight fucan from a starting fucan composition bydialysis, the starting fucan having a broad molecular weightdistribution.

FIG. 8 schematically depicts an exemplary system for obtaining a desiredhigh-molecular-weight fucan from a starting fucan composition using ionadsorption, the starting fucan having a broad molecular weightdistribution.

FIG. 9A depicts NMR results demonstrating that certain fucans treatedaccording to methods herein undergo structural changes to the fucans.

FIG. 9B depicts 2-D NMR results demonstrating that certain fucanstreated according to methods herein undergo chemical structural changesto the fucans.

FIG. 10 shows an exemplary system for the centrifugal precipitation of ahigh-molecular-weight fucan from a starting fucan composition using amulti-segment sucrose barrier, the starting fucan having a broadmolecular weight distribution.

The drawings present exemplary embodiments of the present disclosure.The drawings are not necessarily to scale and certain features may beexaggerated or otherwise represented in a manner to help illustrate andexplain the present systems, methods, etc. Actual embodiments of thesystems, methods, etc., herein may include further features or steps notshown in the drawings. The exemplifications set out herein illustrateembodiments of the systems, methods, etc., in one or more forms, andsuch exemplifications are not to be construed as limiting the scope ofthe disclosure in any manner. The embodiments herein are not exhaustiveand do not limit the disclosure to the precise form disclosed, forexample in the following detailed description.

DETAILED DESCRIPTION

The current compositions, systems, methods, etc., presented hereincomprise high-molecular-weight fucans. The present compositions can beeffective for medical treatments, post-surgical treatments, diseaseinhibition, etc. In some embodiments, the fucan is fucoidan. The presenthigh-molecular-weight fucans can themselves be, or can be included on orin, medical devices, medical materials, combination products or inpharmaceutically acceptable, therapeutically and/or medically effectivecompositions.

The following paragraphs turn to a brief discussion of some of themethodologies that can be used to create the high-molecular-weightfucans and compositions herein from starting fucans and compositions viavarious methods that can be performed using any suitable reactionmixture such as solutions, suspensions, solids, gels or other modalitiesdepending on the chosen method(s).

Compositions

The current compositions, systems, etc., presented herein provide, incertain embodiments, fucans and medically acceptablehigh-molecular-weight fucans and compositions comprising therapeuticallyeffective amounts of high-molecular-weight fucans for the treatment offibrous adhesions, such as surgical adhesions, arthritis, psoriasis orother diseases as desired.

The high-molecular-weight fucans presented herein may be used for aplurality of applications, including the inhibition, prevention,removal, reduction, or other treatment of fibrous adhesions and othertargets and other diseases and/or conditions. Treatment includes thatthe high-molecular-weight fucans reduce or prevent the development of atarget disease or other condition, such as reducing or preventing theformation of fibrous adhesions at a target site, formation of fibrousadhesions at a target site, which is typically a selected target siteidentified by a surgeon or other practitioner as comprising orreasonably susceptible to having fibrous adhesions (or other diseases orconditions), and also includes elimination of existing diseases or otherconditions, including for example the elimination of already-existingfibrous adhesions. For such inhibition, prevention, removal, reduction,or other treatment, the high-molecular-weight fucan is typicallyprovided in a medically acceptable medical device, combination product,or pharmaceutically effective composition that contains additionalcomponents such as binders, adjuvants, excipients, etc., as well as, ifdesired, additional medically active substances such as secondary drugsthat are contained within the composition but not attached to the fucan,and/or that can be attached to the fucan.

The molecular weight distribution of the high-molecular-weight fucansmay be measured using any desired, appropriate measurement system.Different systems can yield different readings or results from differentcompositions having essentially the same make-up, or even from the samebatch when measured differently. One suitable measurement system is anaqueous gel permeation chromatography set up consisting essentially ofone 300 mm analytical gel permeation chromatography column with a 7.8 mminner diameter packed with hydroxylated polymethacrylate-based gel,having an effective molecular weight range of between about 50 kDa andabout 5,000 kDa, one 300 mm analytical gel permeation chromatographycolumn with a 7.8 mm inner diameter packed with hydroxylatedpolymethacrylate-based gel, having an effective molecular weight rangeof between about 1 kDa and about 6,000 kDa and one 40 mm guard columnwith a 6 mm inner diameter packed with hydroxylatedpolymethacrylate-based gel, the two analytical gel permeationchromatography columns and the one guard column contained in a columncompartment at about 30° C., a refractive index detector at about 30°C., 0.1M sodium nitrate mobile phase run at 0.6 mL/min, andquantification against a peak molecular weight standard curve consistingessentially of a first dextran standard with a peak molecular weight ofabout 2,200 kDa, a second dextran standard with a peak molecular weightof between about 720 kDa and about 760 kDa, a third dextran standardwith a peak molecular weight between about 470 kDa and about 510 kDa, afourth dextran standard with a peak molecular weight between about 370kDa and about 410 kDa, a fifth dextran standard with a peak molecularweight between about 180 kDa and about 220 kDa, and a sixth dextranstandard with a peak molecular weight between about 40 kDa and 55 kDa.The peak molecular weight standard curve may further comprise a dextranstandard with a peak molecular weight between 3 kDa and 5 kDa.

The high-molecular-weight fucans herein can have a weight averagemolecular weight over 100 kDa and comprise about 50% w/w or more oftheir molecular weight distribution above 100 kDa. Suchhigh-molecular-weight fucans show greater efficacy in the inhibition,prevention, removal, reduction, and/or other treatment of fibrousadhesions than fucans with weight average molecular weight below 100 kDaand comprising less than about 50% of their molecular weightdistribution above 100 kDa at the same dose. High-molecular-weightfucans with weight average molecular weight above 300 kDa, comprisingabout 70% or more of their molecular weight distribution above 100 kDashow even greater efficacy in the inhibition, prevention, removal,reduction, and/or other treatment of fibrous adhesions at the same dose.

In some embodiments, high-molecular-weight fucans herein are configuredfor use in inhibition, prevention, removal, reduction, or othertreatment of fibrous adhesions that result in greater than about 65%,70%, 80%, 90%, 95%, or 99% efficacious prevention, inhibition or othertreatment of post-surgical adhesions. Such high-molecular-weight fucanscan also be configured for such treatment of other targets.

The high-molecular-weight fucans herein may comprise a molecular weightdistribution in which more than about 60%, 70%, 75%, 80%, 90%, 95 or 99%w/w of the fucan has a molecular weight above 100 kDa.

In other embodiments, the high-molecular-weight fucans herein maycomprise a weight average molecular weight between about 100 kDa and10,000 kDa, between about 140 kDa or 200 kDa and 9,000 kDa, betweenabout 350 kDa or 370 kDa and 8,000 kDa, between about 450 kDa and 7,000kDa, between about 580 kDa and 5,300 kDa or 6,000 kDa, between about 580kDa or 590 kDa and 5,500 kDa, between about 400 kDa and 2,800 kDa orbetween about 800 kDa or 860 kDa and about 2,000 kDa for example about850 kDa, about 930 kDa, about 1,100 kDa, about 1,200 kDa, about 1,300kDa, about 1,600 kDa and about 1,800 kDa.

In yet other embodiments, the high-molecular-weight fucans herein maycomprise a peak molecular weight between about 60 kDa or 70 kDa and7,000 kDa, between about 100 kDa or 140 kDa and 6000 kDa, between about200 kDa or 230 kDa and 5000 kDa, between about 250 kDa and 4000 kDa,between about 350 kDa and 3000 kDa, between about 500 kDa and 2000 kDa,or between about 400 kDa and about 1000 kDa, for example, about 450 kDa,500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa and 750 kDa.

In yet other embodiments, the high-molecular-weight fucans herein maycomprise a number average molecular weight between about 50 kDa and3,000 kDa, between about 100 kDa and 2,000 kDa, between about 200 kDaand 1,500 kDa, between about 300 kDa and 2,000 kDa, between about 400kDa and 1,000 kDa, or between about 250 kDa and about 600 kDa, forexample, about 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa and 550 kDa.

In yet other embodiments, the high-molecular-weight fucans herein maycomprise a molecular weight distribution in which more than about 55%w/w or 60% w/w of the fucan may have a molecular weight above 200 kDa,or more than about 70% w/w or 71% w/w of the fucan may have a molecularweight above 200 kDa. In yet other embodiments, thehigh-molecular-weight fucans herein may comprise a molecular weightdistribution in which more than 22% w/w or 30% w/w of the fucan may havea molecular weight above 500 kDa, or more than 50% w/w or 54% w/w of thefucan may have a molecular weight above 500 kDa.

In yet other embodiments, the high-molecular-weight fucans herein maycomprise a molecular weight distribution in which less than about 10%w/w of the fucan has a molecular weight below 50 kDa, or less than about5% w/w of the fucan has a molecular weight below 50 kDa, or less thanabout 2% w/w of the fucan has a molecular weight below 50 kDa.

In yet other embodiments, the high-molecular-weight fucans herein maycomprise a molecular weight distribution in which less than about 5% w/wof the fucan has a molecular weight below 10 kDa, or less than about 2%w/w of the fucan has a molecular weight below 10 kDa.

In yet other embodiments, the high-molecular-weight fucans herein maycomprise a molecular weight distribution in which less than about 5% w/wof the fucan has a molecular weight below 5 kDa, or less than about 2%w/w of the fucan has a molecular weight below 5 kDa.

In yet another aspect, the high-molecular-weight fucans herein maycomprise a molecular weight distribution in which between 61% w/w and80% w/w or 85% w/w of the fucan has a molecular weight between 200 kDaand 1600 kDa. More particularly, more than 70% w/w of the fucan may havea molecular weight above 200 kDa, and more than 30% of the fucan mayhave a molecular weight above 500 kDa.

In yet another aspect, the high-molecular-weight fucans herein maycomprise a molecular weight distribution in which more than about 20%w/w, 40% w/w or 60% w/w of the fucan has a molecular weight above 1600kDa. More particularly, more than about 70% w/w of the fucan may have amolecular weight above 200 kDa, or more than about 80% w/w of the fucanmay have a molecular weight above 200 kDa.

The high-molecular-weight fucans herein may have a sulfation level ofbetween about 14% w/w and 70% w/w, between about 20% w/w and 60% w/w,between about 30% w/w and 55% w/w, or between about 32% w/w or 35% w/wand 52% w/w.

The high-molecular-weight fucans herein may have a molar ratio of totalfucose:total sulfate of between 1:0.5 and 1:4, between about 1:0.8 and1:3.5, between about 1:1 and 1:2.5, between about 1:1.2 and 1:2.0, orbetween about 1:1.5 and 1:3.

The high-molecular-weight fucans herein may have a molar ratio of totalfucose and galactose:total sulfate of between about 1:0.5 and 1:4,between about 1:0.8 and 1:3.5, between about 1:1 and 1:2.5, betweenabout 1:1.2 and 1:2.0, or between about 1:1.5 and 1:3.

The high-molecular-weight fucans herein may have a total carbohydratecontent of between 27% w/w and 80% w/w, between about 30% w/w and 70%w/w, between about 40% w/w and 90% w/w, or between about 50% w/w and 96%w/w.

The high-molecular-weight fucans herein may have a fucose content as apercentage of total carbohydrate of between about 30% w/w and 100% w/w,between about 40% w/w and 95% w/w, between about 50% w/w and 90% w/w,between about 80% w/w and 100% w/w, or between about 90% w/w and 100%w/w.

The high-molecular-weight fucans herein may have a galactose content asa percentage of total carbohydrate of between about 0% w/w and 60% w/w,between about 3% w/w and 30% w/w, between about 2% w/w and 20% w/w orbetween about 5% w/w and 10% w/w.

The high-molecular-weight fucans herein may have a glucuronic acidcontent as a percentage of total carbohydrate content between about 0%w/w and 10% w/w, a mannose content as a percentage of total carbohydratecontent between about 0% w/w and 7% w/w, a rhamnose content as apercentage of total carbohydrate content between 0% w/w and 4% w/w, anda xylose content as a percentage of total carbohydrate content between0% w/w and 20% w/w. The high-molecular-weight fucans herein may have atotal of glucuronic acid, mannose, rhamnose, glucose and xylose contentas a percentage of the total carbohydrate content below about 30% w/w orbelow about 12% w/w.

In some embodiments, the high-molecular-weight fucans herein, whendissolved at a concentration of about 50 mg/mL in water, have aviscosity of between about 4 cP and about 50 cP, between about 5 cP andabout 40 cP, between about 10 cP and about 30 cP, about 15 cP, about 20cP and about 25 cP. In certain embodiments, the high-molecular-weightfucans herein, when dissolved in water at 1 mg/mL through 100 mg/mL forma solution that is one of clear and colorless, clear and light yellow orclear and light brown.

In certain embodiments, the high-molecular-weight fucans herein can havean acetyl content of less than about 5% w/w, less than about 2% w/w, andabout 0% w/w. In some embodiments, the high-molecular-weight fucansherein comprise substantially 0% w/w acetyl content when measured by 2D¹H-¹³C heteronuclear multiple quantum coherence at 70° C. with solventsignal suppression on a 600 MHz spectrometer equipped with 5-mm coldprobe, in the range from 10-30 ppm in the carbon dimension, in 8increments of 256-512 scans each.

Methods

Methods, systems, etc., are presented for obtaininghigh-molecular-weight fucans obtained from a starting fucan composition,such as a feedstock fucan composition, having a broad molecular weightdistribution (a broad molecular weight distribution starting fucan) thatencompasses and extends beyond the desired high-molecular-weightsegment, the desired high-molecular-weight segment being a portion ofthe broad molecular weight distribution wherein a quantity of the broadmolecular weight distribution at the low molecular weight end has beeneliminated, suppressed or otherwise attenuated. At least one of thesemethods may be used in the preparation of high-molecular-weight fucans,for example, comprising more than about 60%, 70%, 80%, 90% or 95% w/w oftheir molecular weight distribution above 100 kDa. In some embodiments,the current disclosure presents high-molecular-weight fucans that aresuitable for medical and surgical applications, for example, theprevention of surgical adhesions.

Tangential Flow Filtration

Some of the methods discussed herein utilize tangential flow filtration(TFF). Consistent with typical identification of tangential flowfiltration (TFF) filters, the nominal molecular weight cut-off (MWCO)value for a given TFF filter will selectively retain on its retentateside a solution containing molecules that did not cross the filterbarrier and thus generally have molecular weights and/or sizes greaterthan the molecular weight of molecules that do cross/permeate thebarrier to the permeate side. Thus, molecular weight cut-off values forTFF filters are typically not absolute for any given polymer or nominalcut-off value: a given TFF filter will pass or retain some moleculesboth above and below the nominal molecular weight cut-off. The actualcut-off/selectively values and effects of a nominal TFF filter for aparticular polymer can be routinely determined for the particularpolymer.

A number of factors can affect the permeation behavior of the TFFfilters. These factors may be dependent on the TFF filters themselves ordependent on an attribute of the target polymers, for example thefolding behavior and folded structure of the target polymer can affectthe behavior of the target polymer in crossing/not-crossing the TFFfilter's MWCO barrier. Regarding the TFF filters themselves, as isknown, a number of factors can affect the permeation behavior of the TFFfilters. For example, manufacturing methods can cause a variety of holesizes within the specific TFF filter, which variety can include holesboth larger and smaller than the nominal MWCO. Thus, a TFF filter havinga nominal molecular weight cut-off value will substantially pass/retainmolecules at the nominal molecular weight cut-off value, but can alsopass/retain some molecules below and/or above such value.

Gel Permeation Chromatography

Gel permeation chromatography was employed to evaluate the molecularweight distributions obtained for the experimental examples. There are alarge number of different parameters, columns and standards availablefor use in gel permeation chromatography, resulting in a variety ofinstrumentation set-ups available for the analysis of molecular weight.For molecular weight determinations herein, the GPC were conducted usingthe following parameters: The mobile phase was 0.1M sodium nitrate runat 0.6 mL/min. The column compartment and detector were at 30° C. AWaters 2414 refractive index detector was used for detection.

Suitable GPC columns include GPC columns compatible with aqueoussolvents, for example columns packed with at least one of sulfonatedstyrene-divinylbenzene, NH-functionalized acrylate copolymer network,modified silica and hydroxylated polymethacrylate-based gel. For theanalyses herein, three columns were used in series, comprising one 40 mmlong guard column with an inner diameter (ID) of 6 mm packed with 6 μmparticle size hydroxylated polymethacrylate-based gel, followed by afirst 300 mm analytical GPC column with a 7.8 mm ID packed with 12 μmparticle size hydroxylated polymethacrylate-based gel that has anexclusion limit of about 7,000 kDa and an effective molecular weightrange of between about 50 kDa and about 5,000 kDa, followed by a second300 mm analytical GPC column with a 7.8 mm ID packed with 10 μm particlesize hydroxylated polymethacrylate-based gel that has an exclusion limitof about 7,000 kDa and an effective molecular weight range of betweenabout 1 kDa and about 6,000 kDa. The total effective molecular weightrange of the column set up was between about 1 kDa and about 6,000 kDa.An example of this column set up can be Ultrahydrogel®guard-Ultrahydrogel® 2000-Ultrahydrogel® Linear columns connected inseries.

Samples run were quantified against a standard curve comprising oftraceable standards from the American Polymer Standards Corporation:DXT3755K (peak molecular weight=2164 kDa), DXT820K (peak molecularweight=745 kDa), DXT760K (peak molecular weight=621 kDa), DXT670K (peakmolecular weight=401 kDa), DXT530K (peak molecular weight=490 kDa),DXT500K (peak molecular weight=390 kDa), DXT270K (peak molecularweight=196 kDa), DXT225K (peak molecular weight=213 kDa), DXT150K (peakmolecular weight=124 kDa), DXT55K (peak molecular weight=50 kDa), DXT50K(peak molecular weight=44 kDa) and DXT5K (peak molecular weight=4 kDa),the peak molecular weights of these standards being between about 4 kDaand about 2,200 kDa. The standard curve used may, for example, includeDextran 3755 kDa, at least one of Dextran 50 kDa and Dextran 55 kDa, andbetween 3 to 6 additional traceable standards discussed herein, thecalibration points being the peak molecular weights of the calibrantsused. An example calibration curve may consist of DXT3755K, DXT 820K,DXT530K, DXT500K, DXT225K and DXT55K. The columns used herein had atotal effective molecular weight range that encompassed and extendedbeyond the peak molecular weight range of the standards used forquantification of the fucans.

A molecular weight stated for a fucan/fucoidan polymer herein is a valueof molecular weight about which there will always be a distribution ofmolecules of higher and lower molecular weights, increasing ordecreasing in amount or percentage as the molecular weight increases ordecreases away from the specified molecular weight. The distributionmay, but is not required to, have a generally Gaussian or distortedGaussian shape.

Results in the tables herein contain abbreviations used for certaincharacteristics of a molecular weight distribution. Gel permeationchromatography is denoted by GPC, peak retention time is denoted by PRT,peak molecular weight is denoted by PMW, weight average molecular weightis denoted by WAMW, number average molecular weight is denoted by NAMW,percentage distribution is denoted by % dist., molecular weight isdenoted by MW, polydispersity index is denoted by PDI and molecularweight cutoff is denoted by MWCO.

The following paragraphs turn to a brief general discussion of somemethodologies that can be used to create the high-molecular-weightfucans herein.

Sequential Tangential Flow Filtration Segmentation

A high-molecular-weight fucan may be obtained from a broad molecularweight distribution starting fucan composition by a sequential TFFsegmentation method. The methods can comprise: providing a startingfucan composition comprising the desired molecular weight segment, forexample a high-molecular-weight segment, the starting fucan compositionhaving a starting broad molecular weight distribution; subjecting thestarting fucan composition to tangential flow filtration across a first,higher MWCO tangential flow filtration filter having an averagemolecular weight cutoff within the starting molecular weightdistribution; collecting from the first TFF filter a first permeatefucan composition comprising a reduced proportion ofhigh-molecular-weight fucans compared with the starting fucancomposition; subjecting the first permeate fucan composition totangential flow filtration across a second, lower MWCO tangential flowfiltration filter having a lower average molecular weight cutoff withinthe starting molecular weight distribution than the first TFF filter;and, collecting from the second TFF filter a fucan with the desiredmolecular weight segment in the retentate fucan composition.

The methods can comprise further steps as desired, for examplepre-filtering the starting fucan composition through a pre-filtercapable of filtering out particulates or moieties greater than a desiredsize, or other unwanted materials. Passing the starting fucancomposition over the first TFF filter may comprise passing the startingfucan composition over the TFF filter while applying pressure to thestarting fucan composition. Passing the permeate fucan composition ofthe first TFF filter over the second TFF filter may comprise passing thepermeate fucan composition of the first TFF filter over the second TFFfilter while applying pressure to the permeate fucan composition of thefirst TFF filter.

Passing the starting fucan composition over the first TFF filter maycomprise recirculating the retentate fucan composition of the first TFFfilter over the first TFF filter. Recirculating the retentate fucancomposition of the first TFF filter over the first TFF filter maycomprise diafiltering the retentate fucan composition over the first TFFfilter. Recirculating the retentate fucan composition of the first TFFfilter over the first TFF filter may comprise determining a weightaverage molecular weight of the permeate fucan composition of the firstTFF filter. Recirculating the retentate fucan composition of the firstTFF filter over the first TFF filter may comprise recirculating theretentate fucan composition of the first TFF filter over the first TFFfilter until the weight average molecular weight of fucan in thepermeate fucan composition of the first TFF filter has a predetermineddesired value.

Passing a permeate fucan composition from the first TFF filter over thesecond TFF filter may comprise recirculating the permeate fucancomposition over the second TFF filter. Recirculating the permeate fucancomposition over the second TFF filter may comprise diafiltering thepermeate fucan composition over the second TFF filter. Recirculating thepermeate fucan composition over the second TFF filter may comprisedetermining a weight average molecular weight of a retentate fucancomposition of the second TFF filter. Recirculating the permeate fucancomposition over the second TFF filter may comprise recirculating thefucan over the second TFF filter until the weight average molecularweight of the retentate fucan composition of the second TFF filter has apredetermined desired value.

FIG. 1 shows schematically an exemplary molecular weight-basedsegmentation system (higher-to-lower) 100 comprising two different,higher and lower, molecular weight cut-off (MWCO) TFF filters, which inthe embodiment shown are provided as higher molecular weight cut-off TFFfilter 110 and lower molecular weight cut-off TFF filter 120; the TFFfilters can be provided in any acceptable format, the current examplesuse cassettes. Higher molecular weight cut-off TFF filter 110 has a MWCOthat is greater than the MWCO of lower molecular weight cut-off TFFfilter 120. By way of example, higher molecular weight cut-off TFFfilter 110 may have a MWCO of 30 kiloDalton (kDa), 50 kDa, 70 kDa, 100kDa, 300 kDa and 1000 kDa, while the MWCO of lower molecular weightcut-off TFF filter 120 may be, for example, 5 kDa, 10 kDa, 30 kDa, 50kDa and 100 kDa. By way of example, selecting a combination of a highermolecular weight cut-off TFF filter and a lower molecular weight cut-offTFF filter, molecular weight based segmentation system (higher-to-lower)100 can be used to obtain a molecular weight segment between molecularweight cut-off TFF filters of 5-30 kDa, 10-30 kDa, 5-50 kDa, 10-50 kDa,30-50 kDa, 10-70 kDa, 30-70 kDa, 50-70 kDa, 5-100 kDa, 10-100 kDa,30-100 kDa, 50-100 kDa, 70-100 kDa, 5-300 kDa, 10-300 kDa, 30-300 kDa,50-300 kDa, 70-300 kDa and 100-300 kDa. In some embodiments, themolecular weight segment can be a high-molecular-weight segment.

A starting fucan composition is supplied as a solution via input supplyline 102 to higher MWCO subsystem fucan container 116. The startingfucan may be present in a suitable solvent at a concentration between0.1% w/v and 30% w/v, such as between 1% w/v and 10% w/v, for example,at 5% w/v. The starting fucan in a suitable solvent may be pre-filteredthrough pre-filter 104 to remove undesired particulate matter. Thesolution containing the starting fucan composition may comprise furthernon-fucan components such as desired buffers, diluents, etc., asdesired, for example for other fucan processing steps or downstream usesof the fucan. The gauge (effective hole size) of the pre-filter willtypically be greater than the largest polymer molecules to be isolatedby means of the molecular weight based segmentation system(higher-to-lower) 100.

Higher MWCO subsystem pump 114 pumps a solution containing the startingfucan composition to higher molecular weight cut-off TFF filter 110 ofhigher MWCO TFF subsystem 130 via higher MWCO TFF filter supply line112. Higher molecular weight cut-off TFF filter 110 is typicallysupplied as a cassette designed to allow an input fluid to pass over itsfilter on its retentate side. The format of the molecular weight cutofffilter may be without limitation a plate and frame system; a spiralwound cartridge system; a hollow fiber system; a flow cell system; andcentrifugal filter system. The permeate exits via higher MWCO subsystempermeate output line 119 and the treated input fluid, i.e., retentatefluid, leaves as retentate via higher MWCO subsystem retentate returnline 118. Higher MWCO subsystem pump 114 provides a level of pressureover higher molecular weight cut-off TFF filter 110 between itsretentate and permeate sides. In FIG. 1, the retentate fluid from highermolecular weight cut-off TFF filter 110 is returned to higher MWCOsubsystem fucan container 116 via higher MWCO subsystem retentate returnline 118, while permeate fluid is produced via higher MWCO subsystempermeate output line 119 for use outside of the higher MWCO TFFsubsystem 130. While higher MWCO subsystem pump 114 recirculates theprefiltered fucan and retentate over higher molecular weight cut-off TFFfilter 110, solvent may be supplied from higher MWCO subsystem solventcontainer 117 via higher MWCO subsystem solvent supply line 115, forexample to replenish solvent lost via the permeate and/or to ensure thata predetermined number of diavolumes of input starting fucan and solventare circulated over the higher molecular weight cut-off TFF filter 110.

Higher-to-lower MWCO inter-subsystem valve 113 may be shut off (closed)during the above processing, and permeate fluid from higher molecularweight cut-off TFF filter 110 of higher MWCO TFF subsystem 130 can becollected into a container (not shown) for storage or other use beforebeing supplied to lower MWCO subsystem fucan container 126 of lower MWCOTFF subsystem 140. The starting fucan composition can be cycled as manytimes as desired through higher MWCO TFF subsystem 130.

The collected permeate from higher MWCO TFF subsystem 130 may then besupplied to lower MWCO subsystem fucan container 126 of lower MWCO TFFsubsystem 140 via a higher MWCO subsystem permeate output line 119. Inother embodiments, the collected permeate may be transferred in acontainer (not shown) to lower MWCO subsystem fucan container 126. Inyet other embodiments of the system, the higher-to-lower MWCOinter-subsystem valve 113 may be maintained open and the permeate ofhigher molecular weight cut-off TFF filter 110 may be supplied viahigher MWCO subsystem permeate output line 119 on a continuous basis tolower MWCO subsystem fucan container 126. The distribution of highermolecular weight molecules in the permeate of higher molecular weightcut-off TFF filter 110 is attenuated or suppressed compared with thedistribution of higher molecular weight molecules in the starting fucancomposition.

The permeate supplied to lower MWCO TFF subsystem 140 is filtered in asimilar way over lower molecular weight cut-off TFF filter 120 asdiscussed above for higher molecular weight cut-off TFF filter 110. Thatis, after the permeate from higher MWCO TFF subsystem 130 is supplied tolower MWCO subsystem fucan container 126, lower MWCO subsystem pump 124pumps it to lower molecular weight cut-off TFF filter 120 of lower MWCOTFF subsystem 140 via lower MWCO TFF filter supply line 122. Lower MWCOsubsystem pump 124 maintains a level of pressure over lower molecularweight cut-off TFF filter 120 between its retentate and permeate sides.In FIG. 1, the retentate of lower molecular weight cut-off TFF filter120 is returned to lower MWCO subsystem fucan container 126 via lowerMWCO subsystem retentate return line 128, while a permeate is producedvia lower MWCO subsystem permeate output line 129 for further use ordiscarding outside lower MWCO TFF subsystem 140. If the lower MWCOsubsystem pump 124 recirculates the permeate from higher molecularweight cut-off TFF filter 110 and retentate from lower molecular weightcut-off TFF filter 120 to pass again over lower molecular weight cut-offTFF filter 120 (as with the higher molecular weight cut-off filtrationfilter, this recirculation can be repeated as often as desired), solventmay be supplied from lower MWCO subsystem solvent container 127 vialower MWCO subsystem solvent supply line 125 and lower MWCO subsystemfucan container 126 to replenish solvent lost via the lower MWCOsubsystem permeate output line 129 and/or to ensure that a predeterminednumber of diavolumes of retentate of lower molecular weight cut-off TFFfilter 120 and solvent are circulated over the lower molecular weightcut-off TFF filter 120.

During the tangential flow filtration operation of lower MWCO TFFsubsystem 140, lower MWCO subsystem retentate-line valve 106 may beclosed. When the permeate supplied to lower MWCO TFF subsystem 140 fromhigher MWCO TFF subsystem 130 has been filtered to a desired degree,lower MWCO subsystem retentate-line valve 106 is opened and theretentate of lower molecular weight cut-off TFF filter 120 is suppliedvia lower MWCO subsystem retentate output line 108. This provides afucan with the desired molecular weight segment from a starting fucancomposition, for example a high-molecular-weight fucan.

The output fucan has a desired molecular weight segment with a molecularweight distribution typically predominantly between the averagemolecular weight cut-off of the higher molecular weight cut-off TFFfilter 110 and the average molecular weight cut-off of the lowermolecular weight cut-off TFF filter 120. However, considering the widthand complexity of the starting fucan molecular weight distribution andthe variability of polymer behavior and TFF filters, the output polymermolecular weight distribution may not peak between the average molecularweight cut-off values of the two TFF filters. For example, excessivelyhigh or low folding of the fucan can result in selection ofappropriately sized but unusually dense (or not) fucans in the desiredmolecular weight segment. Thus, in terms of the fucans present after thesequential TFF discussed herein, the output desired molecular weightsegment consists essentially of a desired molecular weight segmentderived from the original starting fucan composition that was suppliedto molecular weight based isolation system (higher-to-lower) 100.

Further embodiments can comprise: providing a starting fucan compositioncomprising the desired molecular weight segment, for example ahigh-molecular-weight segment, the starting fucan composition having astarting molecular weight distribution; subjecting the starting fucancomposition to tangential flow filtration across a first tangential flowfiltration filter having an average molecular weight cutoff within thestarting molecular weight distribution; collecting from the first TFFfilter a first retentate fucan composition comprising a reducedproportion of low molecular weight fucans compared with the startingfucan composition; subjecting the first retentate fucan composition totangential flow filtration across a second tangential flow filtrationfilter having a higher average molecular weight cutoff within thestarting molecular weight distribution than the first TFF filter; andcollecting from the second TFF filter a fucan with the desired molecularweight segment in the permeate fucan composition.

The methods may further comprise pre-filtering the starting fucancomposition through a pre-filter capable of filtering out moietiesgreater than a desired size. Passing the starting fucan composition overthe first TFF filter may comprise passing the starting fucan compositionover the first TFF filter while applying pressure to the starting fucancomposition. Passing the retentate fucan composition of the first MCWOfilter over the second TFF filter may comprise passing the retentatefucan composition of the first TFF filter over the second TFF filterwhile applying pressure to the retentate fucan composition of the firstTFF filter in the second TFF filter.

Passing the starting fucan composition over the first TFF filter maycomprise recirculating the retentate fucan composition of the first TFFfilter over the first TFF filter. Recirculating the retentate fucancomposition of the first TFF filter over the first TFF filter maycomprise diafiltering the retentate fucan composition over the first TFFfilter. Recirculating the retentate fucan composition of the first TFFfilter over the first TFF filter may comprise determining a weightaverage molecular weight of the retentate fucan composition of the firstTFF filter. Recirculating the retentate fucan composition of the firstTFF filter over the first TFF filter may comprise recirculating theretentate fucan composition of the first TFF filter over the first TFFfilter until the weight average molecular weight of fucan in theretentate fucan composition of the first TFF filter has a predetermineddesired value.

Passing a retentate fucan composition from the first TFF filter over thesecond TFF filter may comprise recirculating the retentate fucancomposition over the second TFF filter. Recirculating the retentatefucan composition over the second TFF filter may comprise diafilteringthe retentate fucan composition over the second TFF filter.Recirculating the retentate fucan composition over the second TFF filtermay comprise determining a weight average molecular weight of a permeatefucan composition of the second TFF filter. Recirculating the retentatefucan composition over the second TFF filter may comprise recirculatingthe retentate fucan composition over the second TFF filter until theweight average molecular weight of the permeate fucan composition of thesecond TFF filter has a predetermined desired value.

FIG. 2 shows a further embodiment of the methods, systems, etc., herein.In FIG. 2, subsystems 130 and 140 of FIG. 1 are reversed in terms ofprocess order to form molecular weight-based segmentation system(lower-to-higher) 100′. As in the method discussed in FIG. 1, thestarting fucan enters the system through input supply line 102 and ispre-filtered by pre-filter 104. However, in contrast to the method abovein FIG. 1, the pre-filtered starting fucan is processed first in lowerMWCO TFF subsystem 140 then in higher MWCO TFF subsystem 130. In lowerMWCO TFF subsystem 140 the starting fucan composition is passed overlower molecular weight cut-off TFF filter 120, which is the TFF filterwith the lower average MWCO value. In this embodiment, it is theretentate and not the permeate of lower molecular weight cut-off TFFfilter 120 that exits lower MWCO TFF subsystem 140 on lower MWCOsubsystem retentate output line 121. Such retentate exits throughlower-to-higher MWCO inter-subsystem valve 123 to be supplied to higherMWCO subsystem fucan container 116 of higher MWCO TFF subsystem 130. Theretentate is then pumped by higher MWCO subsystem pump 114 via higherMWCO TFF filter supply line 112 to pass over higher molecular weightcut-off TFF filter 110, which is the TFF filter with the higher MWCO.

Within lower MWCO TFF subsystem 140, lower MWCO subsystem pump 124 pumpsthe permeate from lower MWCO subsystem fucan container 126 to lowermolecular weight cut-off TFF filter 120 via lower MWCO TFF filter supplyline 122. In FIG. 2, the retentate of lower molecular weight cut-off TFFfilter 120 is returned to lower MWCO subsystem fucan container 126 vialower MWCO subsystem retentate return line 128, while a permeate isproduced via lower MWCO subsystem permeate output line 129 for furtheruse or discarding outside lower MWCO TFF subsystem 140. If the retentateis recirculated to pass again over lower molecular weight cut-off TFFfilter 120, solvent may be supplied from lower MWCO subsystem solventcontainer 127 via lower MWCO subsystem solvent supply line 125 and lowerMWCO subsystem fucan container 126 to replenish solvent lost via thelower MWCO subsystem permeate output line 129 and/or to ensure that apredetermined number of diavolumes of retentate of lower molecularweight cut-off TFF filter 120 and solvent are circulated over the lowermolecular weight cut-off TFF filter 120.

Lower-to-higher MWCO inter-subsystem valve 123 may be shut during theabove processing, and the retentate of lower molecular weight cut-offTFF filter 120 of lower MWCO TFF subsystem 140 can be collected into acontainer (not shown) before being supplied to higher MWCO subsystemfucan container 116 of higher MWCO TFF subsystem 130. The collectedretentate is supplied to higher MWCO subsystem fucan container 116 ofhigher MWCO TFF subsystem 130 via a physical lower MWCO subsystemretentate output line 121. In other embodiments, the collected retentatemay be transferred in a container (not shown) to higher MWCO subsystemfucan container 116. In yet other embodiments, the lower-to-higher MWCOinter-subsystem valve 123 may be maintained open and the retentate oflower molecular weight cut-off TFF filter 120 supplied via lower MWCOsubsystem retentate output line 121 on a continuous basis to higher MWCOsubsystem fucan container 116. The distribution of lower molecularweight molecules in the retentate from lower molecular weight cut-offTFF filter 120 is attenuated or suppressed compared with thedistribution of lower molecular weight molecules in the starting fucan.

As higher MWCO TFF subsystem 130 processes the retentate from lowermolecular weight cut-off TFF filter 120 of lower MWCO TFF subsystem 140,the permeate of higher molecular weight cut-off TFF filter 110 isproduced on higher MWCO subsystem permeate output line 119. While higherMWCO subsystem pump 114 recirculates the retentate fucan of lower MWCOTFF subsystem 140 over higher molecular weight cut-off TFF filter 110,solvent may be supplied from higher MWCO subsystem solvent container 117via higher MWCO subsystem solvent supply line 115 to replenish solventlost via the permeate and/or to ensure that a predetermined number ofdiavolumes of retentate fucan of lower MWCO TFF subsystem 140 andsolvent are circulated over the higher molecular weight cut-off TFFfilter 110.

In FIG. 2, the retentate fluid from higher molecular weight cut-off TFFfilter 110 is returned to higher MWCO subsystem fucan container 116 viahigher MWCO subsystem retentate return line 118, while permeate fluid isproduced via higher MWCO subsystem permeate output line 119 for useoutside of the higher MWCO TFF subsystem 130. In FIG. 2, the outputfucan with the desired molecular weight segment produced through higherMWCO subsystem permeate output line 119 has a molecular weightdistribution predominantly between the average molecular weight cut-offof the first higher molecular weight cut-off TFF filter 110 and theaverage molecular weight cut-off of the second lower molecular weightcut-off TFF filter 120. However, considering the width and complexity ofthe starting fucan molecular weight distribution and the variability ofpolymer behavior and TFF filters, the output polymer molecular weightdistribution may not peak between the average molecular weight cut-offvalues of the two TFF filters. For example, excessively high or lowfolding of the fucan can result in selection of appropriately sized butunusually dense (or not) fucans in the desired molecular weight segment.Thus, in terms of the fucans present after the sequential TFF discussedherein, the output fucan consists essentially of a desired molecularweight segment of fucan derived from the original starting fucancomposition that was supplied to molecular weight based segmentationsystem (lower-to-higher) 100′. This output fucan with a desiredmolecular weight segment can also be derived from the pre-filteredstarting fucan composition created after prefiltering by pre-filter 104and then supplied to lower MWCO TFF subsystem 140.

Cation Augmented Tangential Flow Filtration

A high-molecular-weight fucan may be obtained from a broad molecularweight distribution starting fucan by cation augmented TFF, the methodscomprising: providing the starting fucan composition having low atomicweight cations and a molecular weight distribution comprising a desiredhigh-molecular-weight segment; cation treating the starting fucancomposition with a cationic additive having cations of greater molecularweight than the low atomic weight cations to replace the low atomicweight cations with additive cations; subjecting the cation-treatedfucan composition to tangential flow filtration across a firsttangential flow filtration filter having an average molecular weightcutoff based on a molecular weight distribution of the desiredhigh-molecular-weight fucan segment to generate a first retentate fucancomposition; subjecting the first retentate fucan composition totangential flow filtration across a second lower MWCO tangential flowfiltration filter having an average molecular weight cutoff based on amolecular weight distribution of the cationic additive to generate asecond retentate fucan composition; subjecting the second retentatefucan composition to diafiltration with a salt solution to generate athird retentate fucan composition; subjecting the third fucan retentatecomposition to diafiltration across the same second tangential flowfiltration filter with a low conductivity diafiltration solution toproduce a fourth retentate fucan composition; and collecting the fourthretentate solution comprising the desired high-molecular-weight fucan.

The methods can comprise further steps as desired, for examplepre-filtering the starting fucan composition through a pre-filtercapable of filtering out particulates or moieties greater than a desiredsize, or other unwanted materials. Passing the starting fucancomposition over the first TFF filter may comprise passing the startingfucan composition over the TFF filter while applying pressure to thestarting fucan composition. Passing the retentate fucan composition ofthe first TFF filter over the second TFF filter may comprise passing theretentate fucan composition of the first TFF filter over the second TFFfilter while applying pressure to the retentate fucan composition of thefirst TFF filter.

Subjecting the first retentate fucan composition to tangential flowfiltration across the second tangential flow filtration filter andtreating the second retentate fucan composition with a salt solution maybe done simultaneously. Treating the second retentate fucan compositionwith a salt may comprise treating the second retentate fucan compositionwith a chloride, bromide, iodide, fluoride, sulfate, sulfite, carbonate,bicarbonate, phosphate, nitrate, nitrite, acetate, citrate, silicateand/or cyanide of an alkali metal, alkaline earth metal, aluminum and/orammonium. Treating the first retentate fucan composition with a sodiumsalt may comprise treating the first retentate with sodium chloride.

Cation treating the starting fucan composition with a cationic additivemay comprise treating the starting fucan with a cationic additive havingcations of greater molecular weight than the low atomic weight cationswithin the starting fucan. The cationic additive may be a polycationicadditive. Cation treating the starting fucan composition with a cationicadditive may comprise treating the starting fucan with a zwitterionicadditive having zwitterions of greater molecular weight than the lowatomic weight cations within the starting fucan.

Subjecting the cation-treated fucan composition to tangential flowfiltration across a first tangential flow filtration filter may compriserecirculating the cation-treated fucan composition over the first TFFfilter. Recirculating the cation-treated fucan composition over thefirst TFF filter may comprise diafiltering the cation-treated fucancomposition over the first TFF filter with a solution of the cationicadditive. Recirculating the cation-treated fucan composition over thefirst TFF filter may comprise determining a weight average molecularweight of fucan in the cation-treated fucan composition. Recirculatingthe cation-treated fucan composition over the first TFF filter maycomprise recirculating the cation-treated fucan composition over thefirst TFF filter until the weight average molecular weight ofcation-treated fucan in the cation-treated fucan composition has apredetermined desired value, producing the first retentate fucancomposition.

Subjecting the first retentate fucan composition to tangential flowfiltration across a second lower MWCO tangential flow filtration filtermay comprise recirculating the first retentate fucan composition overthe second TFF filter. Recirculating the first retentate fucancomposition over the second TFF filter may comprise diafiltering thefirst retentate fucan composition of the second TFF filter with a saltsolution. Recirculating the first retentate fucan composition over thesecond TFF filter may comprise determining a weight average molecularweight of fucan in the first retentate fucan composition. Recirculatingthe first retentate fucan composition over the second TFF filter maycomprise recirculating the first retentate fucan composition over thesecond TFF filter until the weight average molecular weight of fucanfrom the first retentate fucan composition has a predetermined desiredvalue, producing the second retentate fucan composition.

Subjecting the second retentate fucan composition to diafiltration witha salt solution may comprise recirculating the second retentate fucancomposition over the second TFF filter. Recirculating the secondretentate fucan composition over the second TFF filter may comprisediafiltering the second retentate fucan composition of the first TFFfilter with a salt solution comprising at least one of a chloride,bromide, iodide, fluoride, sulfate, sulfite, carbonate, bicarbonate,phosphate and nitrate of an alkali metal, alkaline earth metal, aluminumand ammonium, for example sodium chloride. Subjecting the thirdretentate fucan composition to tangential flow filtration across thesecond MWCO tangential flow filtration filter may comprise recirculatingthe third retentate fucan composition over the second TFF filter.Recirculating the third retentate fucan composition over the second TFFfilter may comprise diafiltering the third retentate fucan compositionof the second TFF filter with a low conductivity solution. The lowconductivity solution may be deionized water.

Cation treating the starting fucan composition with a cationic additivemay comprise treating the input fucan with at least one of choline,polyvinylpyrrolidone, taurine, polyamine, chitosan, histone, andcollagen.

FIG. 3 shows a schematic diagram of a cation-augmented TFF system (CATS)100″ for the separation of a fucan on the basis of molecular weight.CATS 100″ employs a number of elements already discussed at the hand ofFIG. 1 and FIG. 2. A solution containing the starting fucan compositionis supplied via input supply line 102 to higher MWCO subsystem fucancontainer 116. The starting fucan composition in a suitable solvent maybe pre-filtered through pre-filter 104 to remove undesired particulatematter. The solution containing the starting fucan composition maycomprise further non-fucan components such as desired buffers, diluents,etc., as desired, for example for other fucan processing steps ordownstream uses of the fucan. The gauge of the pre-filter will typicallybe greater than the largest polymer molecules to be separated by meansof the CATS 100″.

Cationic additive, for example choline, polyvinylpyrrolidone,polyaniline, may be added to the pre-filtered starting fucan compositionin higher MWCO subsystem fucan container 116. Higher MWCO subsystem pump114 pumps fucan to higher MWCO TFF filter 150 of higher MWCO TFFsubsystem 130′ via higher MWCO TFF filter supply line 112. Higher MWCOTFF filter 150 is typically supplied as a cassette designed to allow aninput fluid supplied to it to pass over its filter on its retentateside, while allowing a permeate to exit via one output line and treatedinput fluid to leave as retentate via another output line. The format ofthe molecular weight cutoff filter may be without limitation a plate andframe system; a spiral wound cartridge system; a hollow fiber system; aflow cell system; and centrifugal filter system. For this embodiment,the cut off molecular weight of higher MWCO TFF filter 150 is chosen toseparate a desired portion of the high-molecular-weight end of thecation-treated fucan obtained by treating the pre-filtered startingfucan with the cationic additive.

Higher MWCO subsystem pump 114 provides a level of pressure over higherMWCO TFF filter 150 between its retentate and permeate sides. In FIG. 3,the retentate of higher MWCO TFF filter 150 is returned to higher MWCOsubsystem fucan container 116 via higher MWCO subsystem retentate returnline 118, while permeate is produced via higher MWCO subsystem permeateoutput line 119 for use outside higher MWCO TFF subsystem 130′ or to bediscarded. While higher MWCO subsystem pump 114 recirculates theprefiltered starting fucan composition and retentate over higher MWCOTFF filter 150, cationic additive flush solution from cationic additiveflush solution container 137 may be supplied via cationic additive flushsolution supply line 135, for example to replenish solution lost via thepermeate on higher MWCO subsystem permeate output line 119 and/or toensure that a predetermined number of diavolumes of input starting fucanand cationic additive flush solution are circulated over the higher MWCOTFF filter 150. By controlling cationic additive flush solution valve136, the cationic additive flush solution may be added in a pulseprocess. In other embodiments, the cationic additive flush solution maybe added in a continuous mode. In other embodiments, the cationicadditive flush solution may be added all at once. If choline has beenchosen as cationic additive for the input starting fucan, then thecationic additive flush solution employed is a choline solution, forexample a choline chloride solution. The number of diavolumes ofretentate and choline flush solution to process over higher MWCO TFFfilter 150 may be predetermined, four diavolumes being a generallyuseful number.

Higher-to-lower MWCO inter-subsystem valve 113 may be shut (closed)during the above processing, and retentate of higher MWCO TFF filter 150of higher MWCO TFF subsystem 130′ collected into a container (not shown)before being supplied to lower MWCO subsystem fucan container 126 oflower MWCO TFF subsystem 140′. The collected retentate may then besupplied to lower MWCO subsystem fucan container 116 of lower MWCO TFFsubsystem 140′ via higher MWCO subsystem retentate output line 111. Inother embodiments, the collected retentate may be transferred in acontainer (not shown) to lower MWCO subsystem fucan container 126. Inyet other embodiments of the system, the higher-to-lower MWCOinter-subsystem valve 113 may be maintained open and the retentate ofhigher MWCO TFF filter 150 may be supplied via higher MWCO subsystemretentate output line 111 on a continuous basis to lower MWCO subsystemfucan container 126. The distribution of lower molecular weightmolecules in the retentate of higher MWCO TFF filter 150 is attenuatedor suppressed compared with the distribution of lower molecular weightmolecules in the starting fucan composition.

The lower MWCO TFF subsystem 140′ removes the choline cations from thecation-treated fucan and restores sodium cations to the fucan, therebyreturning the cation-treated fucan to about its original ioniccomponents, but with a different desired high-molecular-weightdistribution. During the processing of fucan solutions by lower MWCO TFFsubsystem 140′, lower MWCO subsystem output valve 106′ controlling thelower MWCO subsystem retentate output line 108 from lower MWCO subsystemfucan container 126 may be closed. As lower MWCO TFF subsystem 140′processes the retentate from higher MWCO TFF filter 150 of higher MWCOTFF subsystem 130′, the permeate of lower MWCO TFF filter 160 isproduced on lower MWCO subsystem permeate output line 129 via which isemployed elsewhere or is discarded.

While lower MWCO subsystem pump 114 recirculates the retentate of lowerMWCO TFF subsystem 140′ over lower MWCO TFF filter 160, a sodium saltsolution, for example 2M NaCl solution, may be supplied from sodium saltsolution container 142 via sodium salt solution supply line 146 byappropriate control of sodium salt solution control valve 144. For thismethod, the cut off molecular weight of lower MWCO TFF filter 160 ischosen to separate cationic additive released from the fucan by thesodium salt treatment. As the process of lower MWCO TFF subsystem 140′proceeds, the free choline chloride resulting from the replacement ofthe choline cations on the fucan with sodium cations from the NaCltransfers to the permeate of lower MWCO TFF filter 160 and leaves CATS100″ via lower MWCO subsystem permeate output line 129. The sodium saltsolution may be used, for example to replenish solution lost via thepermeate on lower MWCO subsystem permeate output line 129 and/or toensure that a predetermined number of diavolumes of sodium salt solutionand retentate from higher MWCO TFF subsystem 130′ are circulated overthe lower MWCO TFF filter 160. By controlling sodium salt solutioncontrol valve 144, the sodium salt solution may be added in a pulseprocess. In other embodiments, the sodium salt solution may be added ina continuous mode. When a suitable number of diavolumes of sodium saltsolution and retentate have been circulated over lower MWCO TFF filter160, sodium salt solution control valve 144 may be closed and lowconductivity diafiltration solution valve 145 opened. The number ofdiavolumes of sodium salt solution to process over lower MWCO TFF filter160 may be predetermined. Lower-MWCO-subsystem pump 124 provides a levelof pressure over lower-MWCO TFF filter 160 between its retentate andpermeate sides. In FIG. 3, the retentate of lower MWCO TFF filter 160 isreturned to lower MWCO subsystem fucan container 126 via lower MWCOsubsystem retentate return line 128, while permeate is produced vialower MWCO subsystem permeate output line 129 for use outside lower MWCOTFF subsystem 140′ or to be discarded.

Low conductivity diafiltration solution valve 145 may be opened to allowlow conductivity diafiltration solution from low conductivitydiafiltration solution container 143 to enter lower MWCO subsystem fucancontainer 126 via low conductivity diafiltration solution supply line147, the retentate and low conductivity diafiltration solution may beprocessed over lower MWCO TFF filter 160 to remove the free sodium saltgenerated during the sodium salt treatment of the retentate of lowerMWCO TFF filter 160. The low conductivity diafiltration solution may be,for example, deionized water. To this end, the conductivity of permeateon lower MWCO subsystem permeate output line 129 may be measured toensure it drops to a desired level, this serving as indication that thesodium salt has been removed to a suitable degree. The number ofdiavolumes of low conductivity diafiltration solution to process overlower MWCO TFF filter 160 may be predetermined. When the sodium salt hasbeen suitably removed from the retentate of lower MWCO TFF filter 160,low conductivity diafiltration solution valve 145 maybe shut and lowerMWCO subsystem retentate output line 108 opened to deliver the productof CATS 100″ on lower MWCO subsystem retentate output line 108.

Centrifugal Precipitation

A high-molecular-weight fucan may be obtained from a broad distributionstarting fucan by centrifugal precipitation.

Turning to FIG. 4, a centrifugal precipitation system 600 forcentrifugal precipitating a high-molecular-weight fucan from a startingfucan composition is shown. The system 600 comprises a centrifugecontainer 610 comprising a gradated permeable barrier 620. The permeablebarrier may be gradated on the basis of density, with density decreasingfrom a first-bottom end 630 toward a second-top end 640 of thecentrifuge container 610. The gradated permeable barrier 620 may becomprised of different materials of different densities. The gradatedpermeable barrier 620 may be comprised of solutions of differentconcentrations of one solute dissolved in a suitable solvent. Suitablesolvents may be, for example without limitation, one of water and awater-alcohol solution. The solute, also known as “gradient material”may be for example without limitation one or more of glycerol, sorbitol,CsCl, Cs₂SO₄, KBr, diatrizoate, Nycodenz®, iodixanol and suitablesaccharides, including (poly) sucrose. The gradated permeable barrier620 may comprise a continuous gradient of decreasing gradient materialconcentration from the first-bottom end 630 to the second-top end 640 ofthe centrifuge container 610. In other embodiments, the gradatedpermeable barrier 620 may comprise a plurality of distinct gradations indensity, for example gradated permeable barrier segments 620 a, 620 b,and 620 c, as shown in FIG. 4. A solution containing the starting fucancomposition, suitably pre-filtered through a pre-filter to removeparticulate matter, is disposed to be the starting fucan composition 650proximate the second-top end 640 of the centrifuge container 610 and incontact with the gradated permeable barrier 620. The pre-filter may be,for example without limitation, a 0.22 μm particulate filter.

In operation the centrifuge container is subjected to centrifugal forcehaving a force component directed from the second-top end 640 to thefirst-bottom end 630 of the container as indicated by centrifugal forcearrow 660 in FIG. 4. This may be achieved in a suitable centrifuge,schematically shown as centrifuge box 670 in FIG. 4 and adapted toaccommodate the centrifuge container 610. Suitable centrifuges are wellknown in the art and will not be further discussed herein. Thecentrifugal force may be between about 1,000 gravities to about1,000,000 gravities, for example between about 10,000 gravities to about200,000 gravities, between about 60,000 gravities to about 500,000gravities and between about 190,000 gravities to about 800,000gravities.

Associated with the system of FIG. 4, the method for centrifugallyprecipitating a high-molecular-weight fucan from a starting fucancomposition comprises establishing within the centrifuge container 610 agradated permeable barrier 620 of a gradient material having afirst-bottom gradated permeable barrier material end 622 in contact witha first-bottom end 630 of the centrifuge container 610; disposing incontact with an opposing second-top gradated permeable barrier materialend 624 of the gradated permeable barrier 620 proximate a second-top end640 of the centrifuge container 610 the starting fucan compositioncomprising a desired high-molecular-weight segment; subjecting thecentrifuge container 610 to a centrifugal force 660 directed from thesecond-top end 640 to the first-bottom end 630 of the centrifugecontainer 610; and collecting precipitated high-molecular-weight fucanat the first-bottom end 630 of the centrifuge container 610. Disposingthe starting fucan composition 650 in contact with the lowest densitygradient material may comprise pre-filtering the starting fucancomposition through a suitable pre-filter.

Establishing within the centrifuge container 610 a gradated permeablebarrier 620 of a gradient material may comprise establishing a pluralityof segments of gradient material, the density of the gradient materialsegments decreasing from the first-bottom end 630 of the centrifugecontainer 610 toward the second-top end 640 of the centrifuge container610. Establishing within the centrifuge container 610 a gradatedpermeable barrier 620 of a gradient material may comprise establishingwithin the centrifuge container 610 a gradated permeable barrier 620 ofa saccharide. Establishing within the centrifuge container 610 agradated permeable barrier 620 of a gradient material may compriseestablishing within the centrifuge container 610 a gradated permeablebarrier 620 of sucrose. Establishing within the centrifuge container 610a gradated permeable barrier 620 of a gradient material may compriseestablishing within the centrifuge container 610 a gradated permeablebarrier 620 of at least one of glycerol, sorbitol, CsCl, Cs₂SO₄, KBr,diatrizoate, Nycodenz® and iodixanol. Establishing within the centrifugecontainer 610 a gradated permeable barrier 620 of a gradient materialmay comprise establishing within the centrifuge container 610 a gradatedpermeable barrier 620 of a gradient material dissolved in a solvent.Establishing within the centrifuge container 610 a gradated permeablebarrier 620 of a gradient material may comprise establishing within thecentrifuge container 610 a gradated permeable barrier 620 of a gradientmaterial dissolved in one of water and a water-alcohol solution.

FIG. 5 shows another embodiment of a centrifugal precipitation system600′ for centrifugally precipitating a high-molecular-weight fucan froma starting fucan composition. Employing similar numbering as in FIG. 4,this embodiment uses a permeable barrier 620′ having a single barriersegment 620 c′ of gradient material of which a first-bottom permeablebarrier material end 622′ is in contact with a first-bottom end 630 ofthe centrifuge container 610. In this embodiment, the starting fucancomposition is directly in contact with an opposing second-top permeablebarrier material end 624′ of the permeable barrier 620′. In thisembodiment the method comprises subjecting the centrifuge container 610to a centrifugal force 660 directed from the second-top end 640 to thefirst-bottom end 630 of the centrifuge container 610 and collectingprecipitated high-molecular-weight fucan at the first-bottom end 630 ofthe centrifuge container 610. Disposing the starting fucan composition650 in contact with the lowest density gradient material may comprisepre-filtering the starting fucan composition through a suitablepre-filter.

Other embodiments require no barrier to be employed and the containerwith starting fucan composition is centrifuged to subject the centrifugecontainer 610 to a centrifugal force 660 directed from the second-topend 640 to the first-bottom end 630 of the centrifuge container 610 andcollecting precipitated high-molecular-weight fucan at the first-bottomend 630 of the centrifuge container 610.

Gel Electrophoresis-Extraction

A high-molecular-weight fucan may be obtained from a broad molecularweight distribution starting fucan by gel electrophoresis-extraction.The methods can comprise: subjecting the starting fucan compositioncomprising a desired high-molecular-weight-segment to gelelectrophoresis wherein the starting fucan composition is displacedaccording to mass to charge ratio by the action of an applied electricpotential difference; selecting a portion of the electrophoresis gel onthe basis of the potential difference and the desiredhigh-molecular-weight fucan; and extracting the desiredhigh-molecular-weight fucan from the selected gel portion.

Subjecting the starting fucan composition to gel electrophoresis maycomprise first pre-filtering the starting fucan composition in solutionthrough a pre-filter to remove undesired particulate matter. Subjectingthe starting fucan composition to gel electrophoresis may comprisepreparing the starting fucan composition in a solution at aconcentration of between 0.1% w/v and 30% w/v. Extracting the desiredhigh-molecular-weight fucan may comprise extracting the desiredhigh-molecular-weight fucan from a gel portion that extends along the adirection of the potential difference for a distance of between 0.1 mmand 1000 mm. Extracting the desired high-molecular-weight fucan maycomprise extracting the gel portion using one of water, methanol,ethanol, isopropanol, a water/alcohol mix and a salt solution.

Subjecting the starting fucan composition to gel electrophoresis maycomprise displacing the starting fucan composition in solution for apredetermined amount of time. Subjecting the starting fucan compositionto gel electrophoresis across the electrophoresis gel may comprisedisplacing the starting fucan composition across the electrophoresis gelwhile the gel is immersed in a buffer solution. Subjecting the startingfucan composition to gel electrophoresis across the electrophoresis gelmay comprise preparing the gel from a gel material and the buffersolution. Preparing the gel from the gel material and the buffersolution may comprise preparing the gel from the buffer and one ofagarose, polyacrylamide, polydimethylacrylamide and starch. Preparingthe gel from the gel material and the buffer solution may comprisepreparing the gel from one of tris-acetate EDTA, tris-borate EDTA andphosphate buffered saline together with a gel material. Displacing thestarting fucan composition under the action of an applied electricpotential difference may comprise displacing the starting fucancomposition under the action of an applied electric field strength ofbetween about 1 Volt/cm and about 500 Volt/cm, for example between about5 Volt/cm to about 50 Volt/cm, between about 10 Volt/cm to about 200Volt/cm and between about 50 Volt/cm to about 300 Volt/cm.

An electrophoresis-extraction system 900 for obtaining a desiredhigh-molecular-weight fucan from a starting fucan composition is shownin FIG. 6. Electrophoresis-extraction system 900 comprises anelectrophoresis chamber 910, shown as transparent and containingelectrophoresis gel 916, and an electrophoresis buffer 918. Theelectrophoresis gel 916 material may be, for example without limitation,one of agarose, polyacrylamide and a starch. The an electrophoresisbuffer 918 may be for example without limitation one of tris-acetateEDTA, tris-borate EDTA and phosphate buffered saline. Proximate andparallel to a first side of electrophoresis gel 916 is fashioned withinelectrophoresis gel 916 a well 912 in which the starting fucancomposition in solution is placed.

Direct current power supply 920 applies a potential difference acrosselectrophoresis buffer 918 in electrophoresis chamber 910 by means ofcathode 922 and anode 924. The electric potential difference between thecathode 922 and the anode 924 induces the fucan anions in the startingfucan composition to migrate along the gel away from the cathode 922 andtoward the anode 924 along a direction given by migration directionarrow 926 so that, if the potential difference is maintained for a givenperiod of time, different molecular weight molecules of the startingfucan composition will have been displaced from the well 912 bydifferent distances toward the anode 924. The rate of displacement isdetermined by the mass to charge ratio of the fucan molecule. The lowermolecular weight fucans will displace more rapidly and will, after afixed period of time under the action of the electric potentialdifference, be displaced further than the higher molecular weightfucans. Theoretical displacement distances 914 indicate differenttheoretical distances of displacement of different molecular weightfucan molecules, the lower molecular weight fucan molecules beingdisplaced further from the cathode 922 at any given period of time.

To obtain a desired high-molecular-weight fucan from the starting fucancomposition post-electrophoresis, the corresponding portion of theelectrophoresis gel 916 is selected and the high-molecular-weight fucanextracted from that portion of the gel. One non-limiting method of doingthat is to submerge the portion of the electrophoresis gel 916 in anextractant solution and agitate the gel-solution mixture. In oneembodiment, the agitation may be accomplished by shaking. In anotherembodiment, the agitation may be accomplished by high-shear mixing.

Membrane Dialysis

A high-molecular-weight fucan may be obtained from a broad molecularweight distribution starting fucan by membrane dialysis. Consistent withtypical identification of dialysis membranes, the nominal MWCO value fora given dialysis membrane will selectively allow passage of a solutioncontaining molecules generally having molecular weights less than themolecular weight of molecules that do not cross/permeate the dialysismembrane. Molecular weight cut-off values for dialysis membranes aretypically not absolute for any given polymer or nominal cut-off value: agiven dialysis membrane will pass or retain some molecules both aboveand below the nominal molecular weight cut-off. The actualcut-off/selectively values and effects of a nominal MWCO dialysismembrane for a particular polymer can be routinely determined for theparticular polymer.

A number of factors can affect the permeation behavior of the dialysismembranes. These factors may be dependent on the dialysis membranesthemselves or dependent on an attribute of the target polymers, forexample the folding behavior and folded structure of the target polymercan affect the behavior of the target polymer in crossing/not-crossingthe dialysis membrane's MWCO barrier. Regarding the dialysis membranethemselves, for example, manufacturing methods can cause a variety ofhole sizes within the specific dialysis membrane, which variety caninclude holes both larger and smaller than the nominal MWCO cut-off.Thus, a dialysis membrane having a nominal molecular weight cut-offvalue will substantially allow passage of molecules below the nominalmolecular weight cut-off value, but can also pass/retain some moleculesbelow and/or above such value.

The methods can comprise subjecting the starting fucan compositioncomprising a desired high-molecular-weight segment to dialysis against adialysate through a membrane with a molecular weight cut-off greaterthan 100 kDa to produce a dialyzed fucan composition comprising thehigh-molecular-weight fucan; and collecting the dialyzed fucancomposition comprising the high-molecular-weight fucan.

Turning to FIG. 7, a membrane dialysis system 800 for obtaining ahigh-molecular-weight fucan from a starting fucan composition is shown.System 800 comprises a dialysis cell 820 having a dialysis membrane 825that allows low molecular weight fucan molecules to pass through it. Thestarting fucan composition in a suitable solvent enters membranedialysis system 800 and passes into fucan container 810 via input supplyline 801 and through pre-filter 802. The pre-filter may be, for examplea 0.22 μm pre-filter to remove unwanted particulate matter.

The pre-filtered starting fucan composition is circulated through thedialysis cell 820 on a first side of the dialysis membrane 825 by way ofdialysis system supply line 812 and dialyzed fluid return line 816 bydialysis system pump 814. A dialysate fluid is circulated from dialysatecontainer 830 through the dialysis cell 820 on a second side of thedialysis membrane 825 by way of dialysate supply line 832 and dialysatefluid return line 836 by dialysate pump 834. The dialysate fluid isselected to flow freely through the dialysis membrane 825. Suitabledialysate fluids include but are not limited to deionized water andsolutions of sodium chloride, phosphate buffer, sodium phosphate,phosphate buffered saline, tris-HCl buffer, sodium citrate, citratebuffer, sodium ascorbate, ascorbic acid, sodium sulfite andethylenediamine-tetraacetic acid (EDTA). Suitable dialysis membraneshave pore sizes chosen to preferentially stop passage of fucan moleculesof molecular weight greater than 200 kDa. Further suitable dialysismembranes have pore sizes that preferentially prevent the passage ofmolecules of molecular weight greater than 300 kDa, 500 kDa, and 1000kDa. Each of these membranes may be employed to obtain a correspondinghigh-molecular-weight fucan from a starting fucan composition comprisingfewer fucan molecules with molecular weights smaller than the dialysismembrane pore size or cut-off molecular weight relative to the broadstarting molecular weight distribution. The dialysis membrane may be,without limitation, one of a cellulose ester and a regenerated cellulosemembrane. The concentration of the solution containing the startingfucan composition may be between 0.1% w/v and 30% w/v.

As fucan molecules pass through dialysis membrane 825 theirconcentration builds up in the dialysate fluid and this starts to opposethe dialysis process. At a desired point in time dialysate supply valve845 may be opened to allow fresh dialysate fluid into dialysatecontainer 830 from dialysate supply container 840 via dialysate supplyline 842.

After a suitable dialysis period, dialyzed fluid output valve 815 may beopened to allow the dialyzed fucan composition to be drawn from dialysissystem 800 via dialyzed fluid output line 818. Dialysate fluid outputvalve 835 may be opened to allow the dialysis fluid containing lowmolecular weight fucan molecules to be drawn on dialysate fluid outputline 838.

Selective Precipitation

A high-molecular-weight fucan may be obtained from a broad molecularweight distribution starting fucan by selective precipitation. Themethods can comprise: providing the starting fucan compositioncomprising a desired high-molecular-weight segment as a solution of thestarting fucan composition in water; adding to the solution containingthe starting fucan composition a fucan-precipitant to obtain asupersaturated fucan-solvent mix; triggering precipitation of a portionof the broad molecular weight distribution starting fucan by adding anionic-precipitation triggering compound to the supersaturatedfucan-solvent mix to produce a precipitated high-molecular-weight fucanfrom the starting fucan composition and a solution containing remainingfucans; and extracting the precipitated high-molecular-weight fucan fromthe mix. Suitable fucan-precipitants include solvents with a relativepolarity of less than 0.765, for example, ethanol, isopropanol,propanol, acetone, methanol, dimethyl sulfoxide, dimethyl formamide,ethylene glycol, tetrahydrofuran, acetonitrile, glyme, diglyme anddioxane, the solubility of the fucan decreasing as the polarity of theprecipitating fluid decreases. The values for relative polarity can benormalized from measurements of solvent shifts of absorption spectra.See for example Christian Reichardt, Solvents and Solvent Effects inOrganic Chemistry, Wiley-VCH Publishers, 3rd ed., 2003. Suitableionic-precipitation triggering compounds include but are not limited tosalts and bases of monovalent, divalent and trivalent cations, forexample, chlorides, bromides, iodides, fluorides, sulfates, sulfites,carbonates, bicarbonates, phosphates, nitrates, nitrites, acetates,citrates, silicates, hydroxides, oxides and/or cyanides of an alkalimetal, alkaline earth metal, aluminum and/or ammonium. In someembodiments, the ionic precipitation triggering compound comprises atleast one of NaCl, KCl, NaOH, MgCl₂ and CaCl₂). Suitable concentrationsof the starting fucan composition in water are between 0.01% w/v and 30%w/v. Particular fucans lending themselves to the above method includebut are not limited to fucoidan.

The methods may further comprise desalting the starting fucancomposition before adding the fucan-precipitant. The desalting maycomprise diafiltrating the starting fucan composition across a molecularweight cutoff filter. The diafiltrating may comprise diafiltrating thestarting fucan composition with distilled water. The diafiltrating maycomprise diafiltrating the starting fucan composition across a molecularweight cutoff filter having a molecular weight cutoff smaller than adesired molecular weight in the desired high-molecular-weight fucan, forexample, a 5 kDa, 10 kDa, 30 kDa, 50 kDa, 70 kDa, 100 kDa, 200 kDa or300 kDa molecular weight cut-off. The methods may further comprisepre-filtering a solution containing the starting fucan compositionthrough a suitable pre-filter to remove undesired particulate matter.

Extracting the precipitated high-molecular-weight fucan from the mix maycomprise at least one of centrifugation, sedimentation, filtration andhydrodynamic flow separation.

Anionic Adsorption

A high-molecular-weight fucan may be obtained from a broad molecularweight distribution starting fucan by anionic adsorption. The methodscan comprise: providing dissolved in a starting solution, the startingfucan composition having a broad starting molecular weight distributioncomprising a desired high-molecular-weight segment; subjecting thestarting fucan composition in the starting solution to ion exchange withan ion-exchange macroporous resin having a pore size based on a desiredseparation molecular weight within the starting fucan molecular weightdistribution to convert the starting fucan composition into a first ionexchange-treated fucan composition; collecting the first ionexchange-treated fucan composition comprising the desiredhigh-molecular-weight fucan; after the ion exchange with the startingfucan composition subjecting the macroporous resin to a salt solution toextract fucan molecules from the resin into the salt solution, producinga low molecular weight fucan-rich salt solution; desalting the lowmolecular weight fucan-rich salt solution to form a second ionexchange-treated fucan composition; and collecting the second ionexchange-treated fucan composition comprising a low-molecular-weightfucan.

The methods may further comprise desalting the starting fucancomposition before the subjecting to ion exchange. The desalting maycomprise diafiltrating the starting fucan composition across a molecularweight cutoff TFF filter. The diafiltrating may comprise diafiltratingthe starting fucan composition across a molecular weight cutoff TFFfilter having a molecular weight cutoff smaller than a desired molecularweight in the high-molecular-weight fucan, for example a 5 kDa, 10 kDa,30 kDa, 50 kDa, 70 kDa, 100 kDa and/or a 300 kDa molecular weight cutoffTFF filter.

In another embodiment, a method for producing from a starting fucancomposition a desired high-molecular-weight fucan composition, cancomprise: providing dissolved in a starting solution a starting fucancomposition having a broad starting molecular weight distributioncomprising a desired high-molecular-weight segment; subjecting thedissolved starting fucan composition to ion exchange with anion-exchange macroporous resin having a pore size based on a desiredseparation molecular weight within the starting fucan molecular weightdistribution to convert the starting fucan composition into a first ionexchange-treated fucan composition; and collecting the first ionexchange-treated fucan composition comprising the desiredhigh-molecular-weight fucan. The further embodiments may furthercomprise desalting the starting fucan composition before the subjectingto ion exchange. The desalting may comprise diafiltrating the startingfucan composition across a molecular weight cutoff TFF filter. Thediafiltrating may comprise diafiltrating the starting fucan compositionacross a molecular weight cutoff TFF filter having a molecular weightcutoff smaller than a desired molecular weight in a molecular weightdistribution of the desired high-molecular-weight fucan for example a 5kDa, 10 kDa, 30 kDa, 50 kDa, 70 kDa, 100 kDa and/or a 300 kDa molecularweight cutoff TFF filter.

Subjecting the macroporous resin to a salt solution may comprisesubjecting the macroporous resin to a sodium salt solution, for examplea solution comprising at least one of a chloride, bromide, iodide,fluoride, sulfate, sulfite, carbonate, bicarbonate, phosphate, nitrate,nitrite, acetate, citrate, silicate and/or cyanide of an alkali metal,alkaline earth metal, aluminum and/or ammonium. Subjecting themacroporous resin to a sodium salt solution may comprise subjecting themacroporous resin to a sodium chloride solution. Desalting the lowmolecular weight fucan-rich salt solution may comprise diafiltrating thelow molecular weight fucan-rich salt solution across a molecular weightcutoff TFF filter. The diafiltrating may comprise diafiltrating the lowmolecular weight fucan-rich salt solution across a molecular weightcutoff TFF filter having a molecular weight cutoff smaller than adesired molecular weight in a molecular weight distribution of thedesired low molecular weight fucan-rich salt solution for example a 5kDa, 10 kDa, 30 kDa, 50 kDa, 70 kDa and/or 100 kDa molecular weightcutoff TFF filter.

Subjecting the dissolved starting fucan composition to ion exchange withan ion-exchange macroporous resin may comprise adjusting a ratio of thestarting fucan to resin to a predetermined mass ratio. The predeterminedmass ratio may be between about 1:100 fucan:resin and about 10:1fucan:resin, 5:1 fucan:resin, or 2:1 fucan:resin. In other embodiments,the predetermined mass ratio may be between about 1:100 fucan:resin andabout 1:1 fucan:resin. In yet other embodiments, the predetermined massratio may be between about 1:100 fucan:resin and about 1:2 fucan:resin.In yet further embodiments, the predetermined mass ratio may be betweenabout 1:50 fucan:resin and about 1:5 fucan:resin. In yet furtherembodiments, the predetermined mass ratio may be between about 1:20fucan:resin and about 1:1 fucan:resin, for example, about 1:2fucan:resin, 1:4 fucan:resin, 1:6 fucan:resin, 1:8 fucan:resin and 1:10fucan:resin.

Subjecting the dissolved starting fucan composition to ion exchange withan ion-exchange macroporous resin may comprise subjecting the dissolvedstarting fucan composition to ion exchange with the resin for apredetermined period of time. The predetermined period of time may bebetween zero and 300 hours. In other embodiments, the predeterminedperiod of time may be between zero and 100 hours. In furtherembodiments, the predetermined period of time may be between 5 minutesand 30 hours, for example between about 8 hours and about 24 hours. Inyet further embodiments, the predetermined period of time may be between1 and 10 hours, for example between about 4 hours and about 10 hours. Inyet further embodiments, the predetermined period of time may be betweenabout 2 and about 5 hours.

Subjecting the dissolved starting fucan composition to ion exchange withan ion-exchange macroporous resin may comprise subjecting the dissolvedstarting fucan composition to ion exchange with an anion-exchangemacroporous resin. Subjecting the dissolved starting fucan compositionto ion exchange with an anion-exchange macroporous resin may comprisesubjecting the dissolved starting fucan composition to ion exchange witha strong base anion-exchange macroporous resin. Subjecting the dissolvedstarting fucan composition to ion exchange with an anion-exchangemacroporous resin may comprise subjecting the dissolved starting fucancomposition to ion exchange with a weak base anion-exchange macroporousresin. “Strong base” and “weak base” are used according to theirordinary meanings, for example a “strong base” being a resin that doesnot lose charge under any typical ion-exchange circumstances, forexample a quaternary amine functionalized resin, and a weak base being aresin that does lose charge under high pH conditions, for example, aprimary, secondary or tertiary amine functionalized resin. Subjectingthe dissolved starting fucan composition to ion exchange may comprisesubjecting the dissolved starting fucan composition to ion exchange witha mixed charge macroporous resin.

Subjecting the dissolved starting fucan composition to ion exchange withan anion-exchange macroporous resin may comprise subjecting thedissolved starting fucan composition to ion exchange with a macroporousresin comprising at least one of primary, secondary, tertiary andquaternary amine groups. The primary amine groups may be NH₂ groups. Thesecondary amine groups may be at least one of, for example withoutlimitation, benzylamine groups and dimethyl amine groups. The tertiaryamine groups may be at least one of, for example without limitation,diethylaminoethyl groups and dimethylaminoethyl groups. The quaternaryamine groups may be for example without limitation trimethyl ammoniumand triethyl ammonium groups. The resin may comprise, but is not limitedto, one or more of styrene, agarose, dextran, acrylate, methacrylate,methyl methacrylate, butyl methacrylate, divinylbenzene, cellulose,silica, and ceramic.

Subjecting the dissolved starting fucan composition to ion exchange withan ion-exchange macroporous resin may comprise subjecting the dissolvedstarting fucan composition to ion exchange with an ion exchange resinhaving a pore size between 5 nm and 1000 nm, for example between 5 nmand 100 nm, between 10 nm or 15 nm and 50 nm, between 20 nm and 80 nm,between 5 nm and 30 nm, between 100 nm and 500 nm, between 300 nm and900 nm or between 200 nm and 400 nm. Subjecting the dissolved startingfucan composition to ion exchange with an ion-exchange macroporous resinmay comprise subjecting the dissolved starting fucan composition to ionexchange with an ion exchange resin has an exclusion limit of between 50kDa and 50,000 kDa, for example between 50 kDa and 10,000 kDa, between100 kDa and 5,000 kDa, between 10,000 kDa and 40,000 kDa, between 1,000kDa and 9,000 kDa, between 2,000 kDa and 7,000 kDa or between 500 kDaand 2,000 kDa. The exclusion limit can be based on the exclusion limitfor globular proteins.

FIG. 8 shows a schematic diagram of an exemplary ion adsorption system300 for the segmentation of a fucan on the basis of molecular weight. Asolution containing the starting fucan composition is supplied via inputsupply line 301 and pre-filter 306 to TFF subsystem fucan container 176.In a desalting process, tangential flow filtration (TFF) subsystem pump174 pumps the starting fucan composition to TFF filter 171 of TFFsubsystem 170 via TFF subsystem filter supply line 172. The format ofthe TFF filter 171 may be without limitation any one of a plate andframe system; a spiral wound cartridge system; a hollow fiber system; aflow cell system; and centrifugal filter system.

In the system of FIG. 8, TFF subsystem 170 serves as a desalinationsubsystem. TFF filter 171 is typically supplied as a cassette designedto allow an input fluid supplied to it to pass over its filter on itsretentate side, while allowing a permeate to exit via one output lineand treated input fluid to leave as retentate via another output line.For the present method, the cut off molecular weight of TFF filter 171is chosen to allow permeation of salt components in the starting fucansolution while retaining the fucan in the retentate for subsequent ionadsorption treatment in ion exchange subsystem 180. TFF subsystem pump174 maintains a level of pressure over TFF filter 171 between itsretentate and permeate sides. In FIG. 8, the retentate of TFF filter 171is returned to TFF subsystem fucan container 176 via TFF subsystemretentate line 178, while permeate containing the unwanted non-fucansalt components is produced via TFF subsystem permeate output line 179for use outside TFF subsystem 170 or to be discarded.

While TFF subsystem pump 174 recirculates the starting fucan compositionand retentate over TFF filter 171, water or a low conductivity flushsolution from TFF subsystem solvent container 177 may be supplied viaTFF subsystem solvent supply line 175. The flush solution is used toreplenish retentate solution lost via the permeate on TFF subsystempermeate output line 179 and/or to ensure that a predetermined number ofdiavolumes of input starting fucan and solvent are circulated over theTFF filter 171. By controlling TFF subsystem solvent supply valve 173,flush solution may be added in a pulse process. In other embodiments,the solvent may be added in a continuous mode. The continuous mode ofadding the solvent has efficiency benefits. The number of diavolumes ofsolvent to process over TFF filter 171 may be predetermined. In someembodiments, the solvent may be deionized water.

Inter-subsystem valve 302 may be shut during the above processing, andretentate of TFF filter 171 of TFF subsystem 170 collected into acontainer (not shown) before being supplied to ion exchange subsystemfucan container 186 of ion exchange subsystem 180. The collectedretentate may then be supplied to ion exchange subsystem fucan container186 of ion exchange subsystem 180 via TFF subsystem retentate outputline 303. In other embodiments, the collected retentate may betransferred in a container (not shown) to ion exchange subsystem fucancontainer 186. In yet other embodiments of the system, theinter-subsystem valve 302 may be maintained open and the retentate ofTFF filter 171 may be supplied via TFF subsystem retentate output line303 on a continuous basis to ion exchange subsystem fucan container 186.The retentate supplied to ion exchange subsystem 180 may be anticipatedto have a lower salt content remaining that may interfere with theprocessing of fucan in ion exchange subsystem 180 and is a desalinatedfucan composition.

Ion exchange container 181 of ion exchange subsystem 180 contains avolume of macroporous ion exchange resin 189. In some embodiments, themacroporous ion exchange resin is an anion exchange resin. In someembodiments, the macroporous ion exchange resin is a mixed charge resin.The pore size of the macroporous ion exchange resin 189 is chosen topreferentially adsorb fucan molecules of molecular weight below apredetermined value from a solution containing a broad molecular weightdistribution starting fucan, preferentially leaving behind in thesolution fucan molecules that have a greater molecular weight than thepredetermined value. One form of this category of resin is based onsubstantially spherical particles of styrene crosslinked withdivinylbenzene and having pores containing quaternary ammonium groups.In some embodiments, the pore size may be between 10 nm and 100 nm. Thefucan molecules may or may not be preferentially adsorbed into the poresof the resin based on the hydrodynamic size of the fucan molecules.

During the processing of the desalinated fucan composition from TFFsubsystem 170 in ion exchange container 181, ion exchange subsystemoutput valve 304 controlling the ion exchange subsystem output line 305from ion exchange subsystem fucan container 186 may be closed. Ionexchange subsystem salt solution supply valve 183 b and ion exchangesubsystem salt solution return valve 183 c may similarly be closed andion exchange subsystem fucan return valve 183 a opened. While ionexchange subsystem fucan pump 184 a recirculates a solution containingthe desalinated fucan composition through ion exchange container 181 viaion exchange subsystem fucan supply line 182 a and ion exchangesubsystem fucan pump 184 a, macroporous ion exchange resin 189 adsorbsthe lower molecular weight fucan molecules, thereby causing the solutionin ion exchange subsystem fucan return line 188 a to contain the desiredhigh-molecular-weight fucan. After flowing through the ion exchangecontainer 181, the solution containing the desired high-molecular-weightfucan is returned to ion exchange subsystem fucan container 186 via ionexchange subsystem fucan return line 188 a.

The average molecular weight of the fucans in ion exchange subsystemfucan container 186 may be measured or monitored. When the solution inion exchange subsystem fucan container 186 has been circulated for asuitable period of time, or when the fucans in the solution haveattained a predetermined desired average molecular weight value, ionexchange subsystem output valve 304 may be opened to produce a first ionexchange treated fucan composition as the first output product of ionadsorption system 300 via ion exchange subsystem output line 305. Thisfirst output product comprises, for example, a high-molecular-weightfucan with a molecular weight distribution wherein the quantity of theinput starting fucan broad molecular weight distribution at the lowmolecular weight end has been suppressed or attenuated such that theresulting molecular weight distribution is displaced towards the higherend of the molecular weight distribution of the input starting fucancomposition supplied to ion adsorption system 300 on input supply line301.

Ion exchange subsystem output valve 304 may be closed again, as may ionexchange subsystem fucan return valve 183 a, and ion exchange subsystemsalt solution supply valve 183 b and ion exchange subsystem saltsolution return valve 183 c opened to allow salt solution from ionexchange subsystem salt solution container 187 to enter the circulationin ion exchange subsystem 180 via ion exchange subsystem salt solutionsupply line 182 b. Ion exchange subsystem salt solution pump 184 b nowcirculates salt solution via ion exchange subsystem salt solution supplyline 182 b through the macroporous ion exchange resin 189 in ionexchange container 181 and back to ion exchange subsystem salt solutioncontainer 187 via ion exchange subsystem salt solution return line 188 band ion exchange subsystem salt solution return valve 183 c. In thisprocess, the salt displaces the fucan adsorbed within the pores of themacroporous ion exchange resin and releases the freed fucan into thesalt solution in circulation in ion exchange subsystem 180. The saltsolution may be circulated for a predetermined time. In otherembodiments, the average molecular weight of the fucan in the saltsolution in ion exchange subsystem 180 may be measured and therecirculation of the salt solution terminated when the average molecularweight of the fucan in salt solution reaches a predetermined desiredvalue.

In some embodiments, a predetermined amount of a low ionic contentsolution may be used to wash the resin prior to initiating thecirculation of salt solution from ion exchange subsystem salt solutioncontainer 187. In some embodiments, this low ionic content solution maybe deionized water.

At this point ion exchange subsystem output valve 304 may be openedagain and the pumps and valves of ion exchange subsystem 180 suitablyoperated to allow the second product of ion adsorption system 300 drawnfrom ion exchange subsystem output line 305 in the form of a lowmolecular weight fucan-rich salt solution. The second product may befiltered, for example without limitation in a centrifuge over a suitablecentrifugal filter or tangential flow filtration filter, to separate thelow-molecular-weight fucan from the unwanted salt. This produces asecond output low-molecular-weight fucan. This second outputlow-molecular-weight fucan, in contrast with the first outputhigh-molecular-weight fucan discussed above, has a fucan molecularweight distribution wherein a portion of the input starting fucan broadmolecular weight distribution at the high-molecular-weight end has beensuppressed or attenuated such that the resulting molecular weightdistribution is displaced towards the lower end of the molecular weightdistribution of the input starting fucan composition supplied to ionadsorption system 300 on input supply line 301.

Given the width and complexity of the starting fucan molecular weightdistribution and the vagaries of polymer behavior and ion exchangeresins, the two output fucan molecular weight distributions may not peakwhere anticipated from a consideration of the pore size of themacroporous ion exchange resin. If that occurs, however, the two outputfucan molecular weight distributions will still be displaced withrespect to each other, representing the segmentation of the startingfucan composition into a comparatively higher molecular weight fucancorresponding to the first product, and a comparatively lower molecularweight fucan corresponding to the second product. The first productcorresponds to large and heavy fucan molecules preferentially notadsorbed by the resin, while the second product conversely correspondsto fucan molecules preferentially adsorbed by the resin and are onaverage smaller and lighter than those not adsorbed.

Preparative Gel Permeation Chromatography

A high-molecular-weight fucan may be obtained from a broad molecularweight distribution starting fucan by preparative gel permeationchromatography. The methods can comprise providing packed in a columnformat a gel media specified for gel permeation chromatography (GPC) ofpolymers in an aqueous solution; providing a starting fucan compositioncomprising a desired high-molecular-weight segment dissolved in anaqueous solvent suitable for gel permeation chromatography on the gelmedia; subjecting the solution containing the starting fucan compositionto preparative gel permeation chromatography, wherein the fucan isdisplaced according to molecular weight across the gel media in thecolumn at a predetermined flow rate between a first input end of thecolumn and a second output end of the column; collecting eluent from thesecond output end of the column in pre-determined aliquots based on adesired segmentation of the starting fucan composition, each aliquotcomprising a segmented fucan composition; pooling the desired aliquotsbased on the desired segmentation of the starting fucan composition toobtain a pooled GPC aliquot composition comprising the desiredhigh-molecular-weight fucan.

Subjecting the solution containing the starting fucan composition topreparative gel permeation chromatography may comprise firstpre-filtering the starting fucan composition in solution through apre-filter to remove undesired particulate matter. Subjecting thesolution containing the starting fucan composition to preparative gelpermeation chromatography may comprise preparing the starting fucancomposition in a solution at a concentration of between 0.1% w/v and 20%w/v. Subjecting the solution containing the starting fucan compositionto preparative gel permeation chromatography may comprise using at leastone of a peristaltic pump, isocratic pump, binary pump, quaternary pumpand gradient pump to accomplish the displacement across the columncontaining gel media. Subjecting the solution containing the startingfucan composition to preparative gel permeation chromatography maycomprise displacing the solution across the column containing the gelmedia at a predetermined flow rate of between 0.0005 milliliters perminute per gel media surface area (mL/min/cm²) to 5 mL/min/cm², between0.005 mL/min/cm² to 0.5 mL/min/cm², between 0.01 mL/min/cm² to 0.25mL/min/cm², 0.05 mL/min/cm², 0.1 mL/min/cm², 0.15 mL/min/cm² and 0.2mL/min/cm².

Collecting eluent from the second output end of the column may comprisecollecting aliquots of eluent between about 0.1 mL and 1000 mL, betweenabout 1 mL and 100 mL, between about 5 mL and 50 mL, about 10 mL, about20 mL, about 30 mL and about 40 mL. Collecting the aliquots from thesecond output end of the column may comprise measuring the molecularweight distributions of the aliquots by analytical GPC. Measuring thealiquots by analytical GPC may be done simultaneously with thecollecting of the column eluent.

Pooling the desired aliquots may involve measuring the molecular weightdistributions of the aliquots by analytical GPC and pooling onlyaliquots with desired molecular weight distributions. Pooling thedesired aliquots may be done simultaneously with the collecting of thecolumn eluent.

The gel media used may comprise at least one of polyhydroxymethacrylate,sulfonated styrene-divinylbenzene, silica, a hydrophilic bonded phase orpolymer, polystyrene, divinylbenzene, methacrylate, methyl methacrylate,butyl methacrylate, cellulose, ceramic, agarose and dextran. The gelmedia used may have pores with diameters of at least one of about 3 nm,5 nm, 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1,000 nm, 2,000 nm,3,000 nm, 5,000 nm and 10,000 nm. The gel media used may have pores withexclusion limits of at least one of about 100 Da, 100 kDa 1,000 kDa,5,000 kDa, 10,000 kDa, 30,000 kDa, 50,000 kDa and 100,000 kDa. Theexclusion limits may be based on the exclusion limit for globularproteins, or a polysaccharide, for example, dextran and/or pullulan.

The solvent used to dissolve the starting fucan composition may compriseat least one of water, sodium nitrate, lithium nitrate, monosodiumphosphate, disodium phosphate, trisodium phosphate, lithium chloride,lithium bromide, lithium iodide sodium chloride, sodium bromide, sodiumiodide, potassium chloride, potassium bromide, potassium iodide, sodiumhydroxide, lithium hydroxide, potassium hydroxide, sodium sulfate,sodium sulfite, methanol, ethanol and acetonitrile.

Chemical Structural Modification

The methods, systems etc. discussed herein can comprise chemicalstructural modification of the fucan composition, particularly thefucans in the fucan composition. The chemical structural modificationmay involve removal of functional groups from the fucan, for example,0-acetyl, N-acetyl, methoxy, hydroxyl, carboxylic and/or sulfatefunctional groups from the fucan structure. The chemical structuralmodification may involve the use of a wide variety of chemical reagents,for example, acids, bases, detergents and/or oxidizing agents.

Diseases and Conditions

Fibrous Adhesions

A fibrous adhesion is a type of scar that forms between two parts of thebody, usually after surgery (surgical adhesion). Fibrous adhesions cancause severe problems. For example, fibrous adhesions involving thefemale reproductive organs (ovaries, Fallopian tubes) can causeinfertility, dyspareunia and severe pelvic pain. Fibrous adhesions thatoccur in the bowel can cause bowel obstruction or blockage, and fibrousadhesions can also form in other places such as around the heart, spineand in the hand. In addition to surgery, fibrous adhesions can be causedfor example by endometriosis, infection, chemotherapy, radiation, traumaand cancer.

A variety of fibrous adhesions are discussed in this document. Termssuch as surgical adhesions, post-surgical adhesions, postoperativeadhesions, adhesions due to pelvic inflammatory disease, adhesions dueto mechanical injury, adhesions due to radiation, adhesions due toradiation treatment, adhesions due to trauma, and adhesions due topresence of foreign material all refer to adherence of tissues to eachother due to a similar mechanism and are all included in the termfibrous adhesions.

Fibrous adhesion formation is a complex process in which tissues thatare normally separated in the body grow into each other. Surgicaladhesions (also known as post-surgical adhesions) develop from theotherwise normal wound healing response of the tissues to trauma andhave been reported to occur in over two-thirds of all abdominal surgicalpatients (Ellis, H., Surg. Gynecol. Obstet. 133: 497 (1971)). Theconsequences of these fibrous adhesions are varied and depend upon thesurgical site or other site, such as a disease site, involved. Problemsmay include chronic pain, obstruction of the intestines and even anincreased risk of death after cardiac surgery (diZerega, G. S., Prog.Clin. Biol. Res. 381: 1-18 (1993); diZerega, G. S., Fertil. Steril.61:219-235 (1994); Dobell, A. R., Jain, A. K., Ann. Thorac. Surg. 37:273-278 (1984)). In women of reproductive age, fibrous adhesionsinvolving the uterus, fallopian tubes or ovaries are estimated toaccount for approximately 20% of all infertility cases (Holtz, G.,Fertil. Steril. 41: 497-507 (1984); Weibel, M. A. and Majno, G. Am. J.Surg. 126: 345-353 (1973)).

The process of fibrous adhesion formation initially involves theestablishment of a fibrin framework and normal tissue repair. The normalrepair process allows for fibrinolysis alongside mesothelial repair.However, in fibrous adhesion formation the fibrin matrix matures asfibroblasts proliferate into the network and angiogenesis occursresulting in the establishment of an organized fibrous adhesion withinabout 3 to 5 days (Buckman, R. F., et al., J. Surg. Res. 21: 67-76(1976); Raferty, A. T., J. Anat. 129: 659-664 (1979)). Inflammatoryprocesses include neutrophil activation in the traumatized tissues,fibrin deposition and bonding of adjacent tissues, macrophage invasion,fibroblast proliferation into the area, collagen deposition,angiogenesis and the establishment of permanent fibrous adhesiontissues.

Various attempts have been made to prevent surgical adhesions. Theseinvolve pharmacological approaches targeted at influencing thebiochemical and cellular events that accompany surgical traumas well asbarrier methods for the separation of affected tissues. For example, theuse of peritoneal lavage, heparinized solutions, procoagulants,modification of surgical techniques such as the use of microscopic orlaparoscopic surgical techniques, the elimination of talc from surgicalgloves, the use of smaller sutures and the use of physical barriers(films, gels or solutions) aiming to minimize apposition of serosalsurfaces, have all been attempted. Currently, preventive therapies alsoinclude prevention of fibrin deposition, reduction of inflammation(steroidal and non-steroidal anti-inflammatory drugs) and removal offibrin deposits.

Interventional attempts to prevent the formation of post-surgicaladhesions have included the use of hydroflotation techniques or barrierdevices. Hydroflotation involves the instillation of large volumes ofpolymer solutions such as dextran (Adhesion Study Group, Fertil. Steril.40:612-619 (1983)), or carboxymethyl cellulose (Elkins, T. E., et al.,Fertil. Steril. 41:926-928 (1984)), into the surgical space in anattempt to keep the organs apart. Synthetic barrier membranes made fromoxidized regenerated cellulose (e.g., Interceed™),polytetrafluoroethylene (Gore-tex surgical membrane) and fullyresorbable membranes made from a modified hyaluronicacid/carboxymethylcellulose (HA/CMC) combination (Seprafilm™) have alsobeen used to reduce post-surgical adhesion formation in both animals andhumans (Burns, J. W., et al., Eur. J. Surg. Suppl. 577: 40-48 (1997);Burns, J. W., et al., Fertil. Steril. 66:814-821 (1996); Becker, J. M.,et al., J. Am. Coll. Surg. 183:297-306 (1996)). The success of theseHA/CMC membranes may derive from their ability to provide tissueseparation during the peritoneal wound repair process when fibrousadhesions form. The membranes were observed to form a clear viscouscoating on the injured tissue for 3-5 days after application, a timeperiod that is compatible with the time course of post-surgical adhesionformation (Ellis, H., Br. J. Surg. 50: 10-16 (1963)). Unfortunately,limited success has been seen with these methods.

Peritonitis involves inflammation of the peritoneum. Peritonitis cancause severe problems. For example, abdominal pain, abdominal tendernessand abdominal guarding. Peritonitis may involve spontaneous, anatomicand/or peritoneal dialysis related inflammation. Peritonitis may involvean infection, for example, perforation of a hollow viscus, disruption ofthe peritoneum, spontaneous bacterial peritonitis, and systemicinfections may result in infection and peritonitis. Peritonitis may alsonot involve an infection, for example, leakage of sterile body fluidsinto the peritoneum, and sterile abdominal surgery may result inperitonitis. Various attempts have been made to prevent and/or treatperitonitis. For example, general supportive measures such asintravenous rehydration, antibiotics, and surgery. There is an unmetneed for compounds, compositions, methods and the like (includingdelivery approaches) to inhibit, or otherwise treat and/or prevent,peritonitis, preferably more effectively with few side effects.

The high-molecular-weight fucans discussed herein can be used to treatfibrous adhesions in a patient and can be included as a component of, orbe, a high-molecular-weight fucan medical composition, medical device,combination or pharmaceutical product configured and can be composed totreat fibrous adhesions. For example, a high-molecular-weight fucanmedical composition or medical device comprising between about 0.02mg/mL to about 100 mg/mL, for example 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL,0.5 mg/mL, 0.9 mg/mL, 1 mg/mL, 2.5 mg/mL, 5 mg/mL 7.5 mg/mL, of ahigh-molecular-weight fucan herein dissolved in a physiological saltsolution. The physiological salt solution can be, for example, LactatedRinger's Injection USP (LRS), normal saline and physiological Dextransolution.

The high-molecular-weight fucan medical compositions and medicaldevices, which can be liquid medical compositions and devices, hereincan contain pharmaceutically acceptable excipients such as buffers,stabilizers, preservatives, adjuvants, etc. Such high-molecular-weightfucan medical compositions and medical devices can be used to treatfibrous adhesions pre-, during, or post-surgery by administering betweenabout 0.01 mL/kg (per kilogram bodyweight of the patient or target) toabout 10 mL/kg or 15 mL/kg of the fucan medical compositions or devicesin the preceding paragraph. Doses and device quantities include, forexample, about 0.03 mL/kg, 0.1 mL/kg, 0.2 mL/kg, 0.4 mL/kg, 0.5 mL/kg,0.6 mL/kg, 1 mL/kg, 1.2 mL/kg, 2 mL/kg, 3 mL/kg, 4 mL/kg, 5 mL/kg, 8mL/kg, 10 mL/kg and 15 mL/kg of the high-molecular-weight fucan medicalcomposition or medical device to the surgical site of the patient. Infurther embodiments, such high-molecular-weight fucan medicalcompositions and medical devices can be used to treat fibrous adhesionsat any selected target site, for example lesions, abrasions, injurysites, surgical sites and post-surgical sites by administering betweenabout 0.04 mg/kg or 0.1 mg/kg to about 25 mg/kg or 50 mg/kg. Someexamples of such doses include, for example, about 0.04 mg/kg, 0.075mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.3 mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 8 mg/kg, 10 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg and 50 mg/kg of the fucans herein, including for examplethe high-molecular-weight fucans herein, to the surgical site of thepatient. The administering can be accomplished, for example, byinstilling a liquid medical composition or medical device generallythroughout the target area; directing the liquid medical composition ormedical device at a specific location(s) within the target area;spraying the liquid medical composition or medical device generally orat a specific location(s) within the target area; or, spraying orotherwise delivering the liquid medical composition or medical devicevia an applicator, which can be a spray applicator through a trocar,catheter, endoscope or other minimally invasive device, onto a specificlocation(s) that a surgeon or other practitioner has identified asparticularly susceptible to or concerning for development of fibrousadhesions. In another aspect, the administering can be done afteropening of the surgical wound but before the surgical procedure; duringthe surgical procedure, or after the surgical procedure but before thesurgical wound has been closed. If desired, the liquid medicalcomposition or medical device can also be administered after the surgeryis completed (for example through a syringe and needle) and can beadministered to non-surgical target sites as well. The surgical site ofthe patient can be, for example, at least one of the pelvic cavity,abdominal cavity, dorsal cavity, cranial cavity, spinal cavity, ventralcavity, thoracic cavity, pleural cavity, pericardial cavity, skin,joints or muscles. The administering of the high-molecular-weight fucanmedical composition or medical device into the surgical site of thepatient can be accomplished in less than about 15 minutes, 10 minutes, 8minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds and 5seconds.

Examples of administering the high-molecular-weight fucan medicalcomposition or medical device to a surgical site include withoutlimitation administering the high-molecular-weight fucan medicalcomposition or medical device at the surgical site of a Cesarean sectionsurgical procedure; a microvascular free flap reconstruction surgicalprocedure, a full thickness skin graft surgical procedure, a V-Yadvancement flap surgical procedure, a fasciocutaneous rotation flapsurgical procedure, an arthroplasty surgical procedure, a mastectomysurgical procedure, a sequestrectomy surgical procedure, a saucerizationsurgical procedure, an osteotomy surgical procedure, an osteoplastysurgical procedure, a patellectomy surgical procedure, a synovectomysurgical procedure, a capsulectomy surgical procedure, a tendon orligament repair surgical procedure, a tenolysis surgical procedure, atenotomy surgical, a fasciotomy surgical procedure, a meniscal repairsurgical procedure, a vertebrectomy surgical procedure, a ethmoidectomysurgical procedure, a Caldwell Luc's operation surgical procedure, adacryocystorhinostomy surgical procedure, a lysis nasal synechiasurgical procedure, a thymectomy surgical procedure, a pneumonolysissurgical procedure, a pneumonectomy surgical procedure, thoracoplastysurgical procedure, a bilobectomy surgical procedure, a portalhypertension surgery surgical procedure, a splenectomy surgicalprocedure, a esophagectomy surgical procedure, a peritonitis surgerysurgical procedure, a gastrectomy surgery surgical procedure, ajejunojejunostomy surgery surgical procedure, a laparoscopiccholecystectomy surgery surgical procedure, a laparoscopic common bileduct exploration surgical procedure, a gastroenterostomy surgicalprocedure, a bariatric surgery surgical procedure, a bowel resection &anastomosis surgical procedure, a segemental hepatectomy surgicalprocedure, a lobectomy surgical procedure, a pancreatomy surgicalprocedure, a pancreaticoduodenectomy surgical procedure, a tumorresection surgical procedure, a laparoscopic nephrectomy surgicalprocedure, a cystectomy surgical procedure, an abdominal or pelvicadhesion lysis surgical procedure, a hysterosalpingostomy surgicalprocedure, a salpingoplasty surgical procedure, an ectopic pregnancylaparoscopic surgery surgical procedure, a joint replacement surgerysurgical procedure, a broken bone repair surgical procedure, ahysterectomy surgical procedure, a gallbladder removal surgicalprocedure, a heart bypass surgical procedure, an angioplasty surgicalprocedure, an atherectomy surgical procedure, a breast biopsy surgicalprocedure, a carotid endarterectomy surgical procedure, a cataractsurgery surgical procedure, a coronary artery bypass surgical procedure,a dilation and curettage surgical procedure, a hernia repair surgicalprocedure, a lower back pain surgery surgical procedure, a partialcolectomy surgical procedure, prostatectomy surgical procedure and atonsillectomy surgical procedure, after opening the surgical wound,during surgery, before closing the surgical wound and/or after closingthe surgical wound.

Cancers Generally

Cancer has been the second leading cause of death in the U.S. andaccounts for over 20% of all mortalities. Cancer is a proliferativedisease and is characterized by the uncontrolled division of certaincells, which may lead to the formation of one or more tumors. A numberof methods are used to treat cancer, including surgery, radiation,chemotherapy and combinations thereof. Although surgery is a relativelycommon method used for some localized tumors, there is still asignificant chance of tumor recurrence after tumor excision.

Treating cancers and other proliferative diseases has been limited bythe potential for damage or toxicity to non-cancerous, healthy tissues.In radiation and surgical treatments, the procedure has been generallyconfined to and proximal to the tumor sites. However, there can besignificant risk to patients undergoing surgical removal of canceroustissues (e.g., in removal of prostate or brain tumors there can be asignificant risk of non-repairable damage to surrounding vital tissues,for example via potential reduced need for resection of non-tumortissues. Furthermore, in focused radiation treatment, which has beengiven as a first line treatment for prostate cancer, there are similarrisks. In the chemotherapeutic treatment of cancer, the drug has beenadministered systemically, so that the whole body is exposed to thedrug. These drugs are designed to be toxic to cancer cells, but they arealso (generally) toxic to non-cancerous cells so that patients becomequite ill when undergoing drug treatments for cancer. Throughexperience, oncologists are able to give doses of these drugs that maybe tolerated by some patients. However, these doses are often notsuccessful in treating cancers.

One problem with any method of treating cancer has been the localrecurrence of the disease. For example, approximately 700,000 Americansare diagnosed with localized cancer annually (approximately 64% of allcancer patients) and almost half a million are treated using surgicalmethods. Unfortunately, 32% of patients treated with surgery relapseafter the initial treatment (approximately 21% relapse at the initialsurgical site and 11% at distant metastatic sites). Almost 100,000patients die annually due to localized recurrence of cancer. This hasbeen especially true in breast cancer where 39% of patients undergoinglumpectomy will experience local recurrence of the disease.

Staging is a method of judging the progress of the cancer (solid tumor)in a patient. A simplified approach puts patients into three groups orstages based on how far the cancer has advanced:

Stage 1: The cancer can be treated by surgically removing part of theorgan. This is also known as the resectable stage.

Stage 2: The cancer has advanced past the point of being resectable butis still confined to the organ itself.

Stage 3: The tumor has spread to other organs.

Many cancers are treated with anti-proliferative agents including, forexample, 5-fluorouracil (Efudex®), vinca alkaloids (for example,vincristine (Oncovin®)), anthracyclines (for example, doxorubicin(Adriamycin®)), cisplatin (Platinol-AQ®), gemcitabine hydrochloride(Gemzar®), methotrexate and paclitaxel. Some examples of the toxicitiesassociated with the anti-proliferative agents, methotrexate andpaclitaxel, are discussed elsewhere herein. Methotrexate has been usedto treat several cancers including, for example, bladder, breast,cervical, head and neck, hepatic, lung, and testicular cancers.Paclitaxel has been used to treat several cancers including, forexample, ovarian, breast, and non-small cell lung cancers (Compendium ofPharmaceutical and Specialties Thirty-fifth Edition, 2000).

Toxicities due to 5-fluorouracil can include cardiovascular toxicitysuch as myocardial ischemia; central nervous system toxicities such aseuphoria, acute cerebellar syndrome and ataxia; dermatologic toxicitiessuch as alopecia and dermatitis; gastrointestinal toxicities such asnausea, vomiting and oral or gastrointestinal ulceration; hematologictoxicities such as leukopenia, thrombocytopenia and anemia;hypersensitivity toxicities such as anaphylaxis and contacthypersensitivity; ocular toxicities such as increased lacrimation,photophobia and conjunctivitis; and, other toxicities such as fever.5-fluorouracil has been used to treat many cancers including, forexample, breast, colorectal, gastric, hepatic, bladder, head and neck,non-small cell lung, ovarian, pancreatic, and prostate cancers(Compendium of Pharmaceutical and Specialties Thirty-fifth Edition,2000).

Toxicities due to vincristine include central nervous system toxicitiessuch as seizures in children and hallucinations; dermatologic toxicitysuch as alopecia; extravasation toxicity such as vesicant;gastrointestinal toxicities such as nausea, vomiting, constipation andstomatitis; hematologic toxicity such as myelosuppression; neurologictoxicities such as peripheral neuropathy and autonomic neuropathy;ocular toxicities such as double vision, transient blindness and opticatrophy; renal/metabolic toxicities such as urinary retention,hyperuricemia and bladder atony; respiratory toxicity such as shortnessof breath; and, other toxicity such as fever in children. Thisanti-proliferative agent has been used to treat several cancersincluding, for example, Hodgkin's disease, small cell lung, Wilm'stumor, and testicular cancers (Compendium of Pharmaceutical andSpecialties Thirty-fifth Edition, 2000).

Toxicities due to doxorubicin include cardiovascular toxicities such aselectrocardiographic abnormalities and cardiomyopathy; dermatologictoxicities such as alopecia and nail changes; extravasation hazardtoxicity such as vesicant; gastrointestinal toxicities such and nausea,vomiting and stomatitis; genitourinary toxicity such as red colorationof urine; hematologic toxicity such as myelosuppression;hypersensitivity toxicities such as anaphylaxis and skin rash; oculartoxicity such as conjunctivitis; reproductive toxicity such asinfertility; and, other toxicity such as hyperuricemia. Thisanti-proliferative agent has been used to treat several cancersincluding, for example, breast, small cell lung, and ovarian cancers(Compendium of Pharmaceutical and Specialties Thirty-fifth Edition,2000).

Toxicities due to cisplatin include cardiovascular toxicity such aselectrocardiographic changes; dermatologic toxicity such ashyperpigmentation; extravasation hazard toxicity such as irritant;gastrointestinal toxicities such as nausea and vomiting; hematologictoxicities such as myelosuppression and hemolytic anemia;hypersensitivity toxicity such as anaphylactic; neuromuscular toxicitysuch as peripheral neuropathy and acute encephalopathy; ocular toxicitysuch as retrobulbar neuritis; otologic toxicities such as hearing lossand tinnitus; renal/metabolic toxicities such as toxic nephropathy andhypokalemia; and, other toxicity such as infertility. Thisanti-proliferative agent has been used to treat several cancersincluding, for example, bladder, small cell lung, ovarian, testicular,brain, breast, cervical, head and neck, hepatoblastoma, and thyroidcancers (Compendium of Pharmaceutical and Specialties Thirty-fifthEdition, 2000). Toxicities due to gemcitabine hydrochloride include, forexample, hematologic toxicities such as myelosuppression;gastrointestinal toxicities such as nausea, vomiting and stomatitis;hepatic toxicities such as transient elevations of serum transaminases;renal toxicities such as proteinuria, hematuria, hemolytic uremicsyndrome and renal failure; dermatologic toxicity such as rash andalopecia; edema toxicities such as edema and peripheral edema; and,other toxicity such as fever. This anti-proliferative agent has beenused to treat pancreatic and non-small cell lung cancers (Compendium ofPharmaceutical and Specialties Thirty-fifth Edition, 2000).

The present discussion comprises prevention or treatment of localizedcancers or solid tumors that can be treated include those of theprostate, breast, pancreas, liver, kidney, genitourinary system, brain,gastrointestinal system, respiratory system, and head and neck. Thecompositions, etc., herein may prevent or treat cancers, includingmetastases, by allowing controlled release of high-molecular-weightfucan at a site somewhat distant from the target tumors by allowingeffective concentrations of the high-molecular-weight fucan to reach thetumors and/or metastases by diffusion or even systemic transport. Someof these cancers are discussed further in the following paragraphs.

Prostate Cancer

Prostate cancer is a malignant tumor that arises in the cells lining theprostate gland. In the U.S., an estimated 200,000 patients will developprostate cancer this year, and more than 30,000 will die of the disease.Prostate cancer has a death to new cases ratio of ˜15%. The cancer mayremain within the prostate, or it may spread to surrounding tissues orto distant sites (most often lymph nodes and bone). Usually prostatecancer spreads silently, producing symptoms only when it has progressedbeyond the prostate. If prostate cancer has been diagnosed and treatedduring early stages, in some studies patients have had a 5-year survivalrate of 94%.

Prostate cancer is often discussed as a disease of men over age 50. Infact, 80% of men with prostate cancer are 60 years of age and older. Aman's chances of being diagnosed with prostate cancer during hislifetime are about 1 in 10, roughly the same as a woman's chances ofhaving breast cancer. The number of reported new cases has risendramatically in recent years as a result of improved tests that candetect the disease early in its development, often long before symptomsappear. The likelihood of developing prostate cancer in any given yearincreases with age but rises dramatically after age 50.

Current treatment options for prostate cancer depend upon the extent ofdisease progression, the patient's age and overall health. Elderlypatients, who have only early stage cancer or who suffer fromadditional, more serious diseases, may be treated conservatively,whereas those whose cancer is advanced may undergo more aggressivetreatment. Prostate cancer has been treated by various methods,including radiation therapy (external beam radiation or brachytherapy),hormone withdrawal or castration (surgical or chemical),anti-proliferative agents, surgery, and expectant therapy (that is,“watchful waiting”). No treatment guarantees an absolute cure, and somehave considerable side effects.

Early stage prostate cancer (that is, the tumor is localized to theprostate) may be treated with “watchful waiting”. Surgery for prostatecancer has been recommended for patients whose overall health has beenotherwise good and the tumor is confined to the prostate gland. A commontreatment for localized cancer of the prostate in men under the age of70 has been radical prostatectomy (that is, surgical removal of theprostate).

Patients whose cancer is localized in the prostate area are commonlytreated with external beam radiation (EBR). The radiation kills cancercells and shrinks tumors. EBR accounts for less than 20% of localizedprostate cancer treatment, with approximately 50% of these patientsexperiencing post radiation recurrences of the disease. Combined withearly stage prostate cancer detection and increased demand frompatients, brachytherapy (i.e., local radiation therapy) use has beenexpected to grow. In 1995, only 2.5% of newly diagnosed patients weretreated using brachytherapy. Brachytherapy involves the implantation ofradioactive metal “seeds” in the prostate tumor.

Treatment for prostate cancer that has spread involves removal of thetesticles or hormone therapy. Both are used to inhibit or stop theproduction of the testosterone that has been driving the cancer growth.Approximately 20% of all prostate cancer patients undergo hormonewithdrawal therapy. Hormone therapies include goserelin acetate(Zoladex®) or leuprolide acetate (Lupron®). Anti-proliferative agentsused to treat prostate cancer have included 5-fluorouracil.

Breast Cancer

In the U.S., breast cancer has been the most common cancer among women,with about 180,000 new cases diagnosed every year (male breast canceraccounts for about 5% of all diagnosed breast cancers). It has beensurpassed only by lung cancer as a cause of death in women, and it hasbeen responsible for approximately 50,000 deaths annually. An Americanwoman has a one in eight (or about 13%) chance of developing breastcancer during her lifetime. Over the past decade, most reported breastcancers were small, primary (arising independently; not caused by ametastasis) tumors. Roughly 70% to 80% of newly diagnosed patientsexhibited early-stage disease (Stage 1 or 2), and a majority had noinvolvement of the axillary (underarm) lymph nodes.

Most breast cancers are carcinomas (that is, malignant tumors that growout of epithelial tissues). Less than 1% of breast cancers are sarcomas,or tumors arising from connective tissue, bone, muscle or fat. Inaddition, most breast cancers (about 75%) are ductal carcinomas, arisingin the tissues that line the milk ducts. A much smaller number ofcancers (about 7%) are found within the breast lobules and are calledlobular carcinomas. Paget's disease (cancer of the areola and nipple)and inflammatory carcinoma account for nearly all other forms of breastcancer.

Breast cancer treatment has been complicated and depends on manyfactors. Two important factors are the type of tumor and the stage ofprogression. Tumor characteristics, in particular, help to separateindividuals into two groups: (1) those who are at low risk of cancerrecurrence and (2) those who are at high risk of cancer recurrence.Specific prognostic factors place patients in either of these groups.These factors include tumor size; presence of female sex hormoneestrogen and progesterone (ER/PR) receptors; cellular growth cycle phase(whether tumor cells are actively dividing or are in “S-phase”);presence of a protein known as “her-2-neu protein”; tumor grade, anindicator of tumor cell differentiation or change; and, tumor ploidy,the number of sets of genetic material within tumor cells.

Treatment of primary disease without significant lymph node involvementhas been by lumpectomy and radiotherapy. More significant lymph nodeinvolvement may warrant mastectomy and removal of auxiliary lymph nodes.At this stage the chance of metastasis and local recurrence has beenhigh. Treatment of metastatic disease has been palliative, involvingradiation therapy and chemotherapy, which are immunosuppressive,cytotoxic and leukopenia. Anti-proliferative agents including, forexample, 5-fluorouracil, doxorubicin, methotrexate, and paclitaxel, havebeen approved for use against breast cancer.

Pancreatic Cancer

The pancreas is an organ of the digestive system located near thestomach and small intestine. It has two major functions: the productionof enzymes and hormones. Cancers of the pancreas can occur in theexocrine (i.e., enzymes) pancreas (e.g., classic pancreaticadenocarcinomas) or can occur in the endocrine (i.e., hormones)pancreas.

Cancers of the exocrine pancreas are a very serious health issue. In theU.S., approximately 28,000 patients are diagnosed with pancreaticcancer, while about the same number die annually from this disease.Pancreatic cancer occurs equally in males and females. Due todifficulties in diagnosis, the intrinsic aggressive nature of pancreaticcancers, and the sparse systemic treatment options available, onlyapproximately 4% of patients diagnosed with pancreatic adenocarcinomalive for 5 years after diagnosis. Pancreatic cancer has been the 5^(th)leading cause of cancer death, following breast, lung, colon, andprostate cancer.

The choice of treatment for pancreatic cancer depends largely on thestage of the tumor. Possible treatments include surgery,anti-proliferative agents, radiation, and biological therapy. Surgeryhas been usually reserved for Stage 1 patients whose cancer is deemedresectable. Sometimes a combination of therapies, such as radiation andanti-proliferative agent given before or after surgery, can increase apatient's chances of survival. Pancreatic cancer that is deemedunresectable (usually Stage II or later) may be treated usinganti-proliferative agents in clinical trials. Anti-proliferative agents,such as, for example, gemcitabine or 5-fluorouracil have had some effectagainst pancreatic cancer and gemcitabine has been used as a palliativeagent. Toxicities due to these anti-proliferative agents are discussedelsewhere herein. Radiation therapy has some effect against pancreaticcancer when used in combination with chemotherapy. Radiation therapyalone may subdue symptoms. This form of treatment has also been used inStage II or later pancreatic cancers.

Bladder Cancer

In 1998, it was estimated that over 54,000 new cases of bladder cancerwould be diagnosed in the U.S. and about 15,000 deaths would beattributed to the disease. Bladder cancer has been the fourth mostcommon cancer among American men and the ninth most common cancer amongAmerican women. It occurs three times more frequently in men than inwomen. Primarily a disease of older men, bladder cancer has been asignificant cause of illness and death. The risk of bladder cancerincreases steeply with age (80% of cases occur in people older than 50years), with over half of all bladder cancer deaths occurring after age70. In white men over 65, the annual disease rate of bladder cancer hasbeen approximately 2 cases per 1,000 persons; this contrasts with a rateof 0.1 cases per 1,000 persons under 65. During one's lifetime, theprobability of developing bladder cancer has been greater than 3%;however, the probability of dying, from bladder cancer has been small(<1%). Bladder cancer rarely occurs in people who are younger than 40years of age.

Recent studies suggest that certain genes and inherited metabolicabilities may play a role in bladder cancer. Transitional cell carcinoma(TCC) has been the most common form of bladder cancer. TCC usuallyoccurs as a superficial (surface), papillary (wart-like), exophytic(outward-growing) mass upon a stalk-like base. In some cases, though,TCC may be attached on a broad base or it may appear ulcerated (withinan indented lesion). Papillary TCCs often start out as areas ofhyperplasia that later dedifferentiate or lose individual cellcharacteristics. Only about 10% to 30% of papillary TCCs develop intoinvasive cancers. By contrast, nonpapillary forms of TCC are more likelyto become invasive. As noted, such TCCs may appear ulcerated or flat.Flat, nonpapillary TCC that has been made up of anaplastic epitheliumhas been classified as carcinoma in situ (CIS or TIS). The tissue of CIScontains cells that are large, have noticeable nucleoli (round bodywithin a cell; involved in protein synthesis), and lack normal polarity.

The treatment of bladder cancer depends upon many factors. The mostimportant of these factors are the type of tumor that is present and itsstage. Common treatments include transurethral resection (TUR),electrosurgery, laser surgery, intravesical therapy, anti-proliferativeagents, surgical therapy, cystectomy, and radiation therapy. Examples ofanti-proliferative agents used to treat bladder cancer include, forexample, 5-fluorouracil, cisplatin and methotrexate. Toxicities due tothe anti-proliferative agents, 5-fluorouracil, cisplatin, andmethotrexate, are discussed elsewhere herein.

Brain Cancer

Brain tumors are often inoperable and more than 80% of patients diewithin 12 months of diagnosis. Approximately 18,000 new cases of primaryintracranial (brain) cancer are diagnosed each year in the U.S. Thisrepresents about 2 percent of all adult cancers. More than 50 percent ofthese are high-grade gliomas (i.e., glioblastoma multiform andanaplastic astrocytoma tumors). Patients with these tumors often sufferfrom severe disabilities such as motor dysfunction, seizures, and visionabnormalities.

Tumors that begin in brain tissue are known as primary brain tumors.Primary brain tumors are classified by the type of tissue in which theybegin. The most common brain tumors are gliomas, which begin in theglial (supportive) tissue. Others include astrocytomas, brain stemgliomas, ependymomas and oligodendrogliomas.

Surgical removal of brain tumors has been recommended for most types andin most locations and should be as complete as possible within theconstraints of preservation of neurologic function. An exception to thisrule has been for deep-seated tumors, such as pontine gliomas, which arediagnosed on clinical evidence and are treated without initial surgeryapproximately 50% of the time. In many cases, however, diagnosis bybiopsy is performed. Stereotaxic biopsy can be used for lesions that aredifficult to reach and resect. Patients who have brain tumors that areeither infrequently curable or unresectable should be consideredcandidates for clinical trials that evaluate radiosensitizers,hyperthermia, or interstitial brachytherapy used in conjunction withexternal-beam radiation therapy to improve local control of the tumor orfor studies that evaluate new drugs and biological response modifiers.

Radiation therapy has a major role in the treatment of most tumor typesand can increase the cure rate or prolong disease-free survival.Radiation therapy may also be useful in the treatment of recurrences inpatients treated initially with surgery alone. Chemotherapy may be usedbefore, during, or after surgery and radiation therapy. Recurrent tumorsare treated with chemotherapy as well. Anti-proliferative agents used inthe treatment of brain cancers include cisplatin. Examples of thetoxicities associated with this anti-proliferative agent are discussedelsewhere herein.

Restenosis

Restenosis is a form of chronic vascular injury leading to vessel wallthickening and loss of blood flow to the tissue supplied by the bloodvessel. This inflammatory disease can occur in response to vascularreconstructive procedures including any manipulation that relievesvessel obstruction. Thus, restenosis has been a major restrictive factorlimiting the effectiveness of these procedures.

The present discussion comprises prevention or treatment of restenosis,for example by administering to a blood vessel a therapeuticallyeffective amount of the combination of an oligonucleotide therapeuticand an anti-inflammatory agent. Suitable compositions include apolymeric carrier that can be surgically implanted at a restenosis site,or potential restenosis site, or can be injected via a catheter as apolymeric paste or gel. Suitable compositions may comprisehigh-molecular-weight fucans discussed herein.

Arthritis

Rheumatoid arthritis (RA) is a debilitating chronic inflammatory diseasecharacterized by pain, swelling, synovial cell proliferation (pannusformation) and destruction of joint tissue. In the advanced stage, thedisease often damages critical organs and may be fatal. The diseaseinvolves multiple members of the immune system (macrophages/monocytes,neutrophils, B cells and T cells) complex cytokine interactions andsynovial cell malfunction and proliferation. Early aggressive treatmenthas been recommended with disease modifying anti-rheumatic drugs(DMARDs) such as methotrexate, which drug is discussed elsewhere herein.

Crystal induced arthritis has been characterized by crystal inducedactivation of macrophages and neutrophils in the joints and is followedby excruciating pain for many days. The disease progresses so that theintervals between episodes gets shorter and morbidity for the patientincreases. This disease has been generally treated symptomatically withnon-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac sodium(Voltaren®). This anti-inflammatory agent has toxicities which includecentral nervous system toxicities such as dizziness and headache;dermatologic toxicities such as rash and pruritus; gastrointestinaltoxicities such as exacerbated ulcerative colitis and Crohn's disease;genitourinary toxicities such as acute renal failure and renal papillarynecrosis; hematologic toxicities such as agranulocytosis, leukopenia andthrombocytopenia; hepatic toxicities such as elevated livertransaminases and hepatitis; and, other toxicities such as asthma andanaphylaxis.

The present discussion comprises prevention or treatment of rheumatoidarthritis, for example via administering to a patient a therapeuticallyeffective amount of an oligonucleotide therapeutic and optionally ananti-inflammatory agent. Suitable compositions include a polymericcarrier that can be injected into a joint as a controlled releasecarrier of the anti-inflammatory agent and microparticulates ascontrolled release carriers of the oligonucleotide therapeutic (which inturn has been incorporated in the polymeric carrier). Suitablecompositions may comprise high-molecular-weight fucans discussed herein.Such polymeric carriers may take the form of polymeric microspheres,pastes or gels.

Inflammatory Conditions

The compositions, etc., herein may optionally inhibit or treatinflammatory conditions involving neutrophils for example comprisingadministering to a patient compositions containing an oligonucleotidetherapeutic and an anti-inflammatory agent. Examples of such conditionsinclude crystal-induced arthritis; osteoarthritis; non-rheumatoidinflammatory arthritis; mixed connective tissue disease; Sjögren'ssyndrome; ankylosing spondylitis; Behçet's syndrome; sarcoidosis;psoriasis; eczema; inflammatory bowel disease; chronic inflammatory lungdisease; neurological disorders; and, multiple sclerosis. Some of thesediseases are discussed further in the following paragraphs.

Chronic Inflammatory Skin Diseases (Including Psoriasis and Eczema)

Psoriasis is a common, chronic inflammatory skin disease characterizedby raised, thickened and scaly lesions which itch, burn, sting and bleedeasily. While these diseases have cellular proliferation and angiogeniccomponents in later stages of the disease, patients often haveaccompanying arthritic conditions. Symptoms may be treated withsteroidal anti-inflammatory agents such as prednisone oranti-proliferative agents such as methotrexate, which agents arediscussed elsewhere herein. The compositions herein may also be used toinhibit or otherwise treat and/or prevent chronic inflammatory skindiseases, for example psoriasis and/or eczema.

The following provides some additional representative examples ofinflammatory diseases that can be treated with compositions discussedherein, include, for example, arterial embolization in arteriovenousmalformations (vascular malformations); menorrhagia; acute bleeding;central nervous system disorders; and, hypersplenism; inflammatory skindiseases such as psoriasis; eczematous disease (atopic dermatitis,contact dermatitis, eczema); immunobullous disease; and, inflammatoryarthritis which includes a variety of conditions including rheumatoidarthritis, mixed connective tissue disease, Sjögren's syndrome,ankylosing spondylitis, Behçet's syndrome, sarcoidosis, crystal inducedarthritis and osteoarthritis (all of which feature inflamed, painfuljoints as a prominent symptom).

Ischemia

Ischemia or ischaemia involves a restriction in blood supply, which mayinclude a shortage of supply of oxygen, glucose and other componentsrequired for proper tissue function, resulting in damage and/ordysfunction of tissue. Ischemia can cause severe problems. For example,tissues can become anoxic, necrotic, and clots can form. Variousattempts have been made to prevent and/or treat ischemia. For example,restoration of blood flow, or reperfusion. Restoration of blood,however, involves the reintroduction of oxygen, which can causeadditional damage due to the production of free radicals, resulting inreperfusion injury. Reperfusion injury can cause severe problems. Thecompositions herein may be used to inhibit or otherwise treat and/orprevent, ischemia, and/or reperfusion injury.

Endotoxemia

Endotoxemia is the presence of endotoxins in the blood. Endotoxemia cancause severe problems. For example, endotoxemia can lead to septicshock. The compositions herein may be used to inhibit, or otherwisetreat and/or prevent, endotoxemia.

Keloid Scarring

Keloid trait causes wounds to heal with raised scars. Keloid traits'raised scars involve abnormal fibrous scarring. Keloid trait causessevere problems, for example, pain and disfigurement. The compositionsherein may be used to inhibit, or otherwise treat and/or prevent, keloidtrait and its resulting raised scars.

Keloid (keloid scar) is a type of scar that expands in growths overnormal skin. Keloids involve abnormal collagen growth, including type Iand type III collage abnormal growth. Keloids cause severe problems, forexample, pain, itchiness, and if infected may ulcerate. Attempts havebeen made to treat or prevent keloids including the use of surgery,dressings, steroid injections and laser therapy. The compositions hereinmay be used to inhibit, or otherwise treat and/or prevent, keloids.

Dermatitis

Dermatitis includes inflammation of the skin including atopic dermatitisand contact dermatitis. For example, contact dermatitis involveslocalized rash and/or irritation of the skin following contact of theskin with a foreign substance. For example, atopic dermatitis is achronically relapsing, pruritic skin disease. Atopic dermatitis issometimes called prurigo Besnier, neurodermitis, endogenous eczema,flexural eczema, infantile eczema, childhood eczema and prurigodiathsique. Eczema is a disease in a form of dermatitis. Other types ofdermatitis include spongiotic dermatitis, seborrhoeic dermatitis(dandruff), dyshidrotic dermatitis (pompholyx), urticaria, vesiculardermatitis (bullous dermatitis), and popular urticaria. Dermatitis cancause severe problems. For example, dry skin, skin rashes, skin edema,skin redness, skin itchiness, skin crusting, cracking, blistering,oozing and bleeding. Attempts have been made to treat or preventdermatitis including the use of corticosteroids and coal tars. Thecompositions herein may be used to inhibit, or otherwise treat and/orprevent, dermatitis including atopic dermatitis, eczema, contactdermatitis, spongiotic dermatitis, seborrhoeic dermatitis, dyshidroticdermatitis, urticaria, vesicular dermatitis, and popular urticaria.

Rosacea

Rosacea is a chronic disease or condition typically characterized byfacial erythema. Rosacea can cause severe problems. For example, rosaceatypically begins as redness on the forehead, nose or cheeks and can alsocause redness on the neck, ears, scalp and chest. For example, rosaceacan cause additional symptoms including telangiectasia, papules,pustules, painful sensations, and in advanced cases rhinophyma (redlobulated nose) may develop. Rosacea subtypes includeerythematotelangiectatic rosacea, papulopustular rosacea, phymatousrosacea, and ocular rosacea. Attempts have been made to treat or preventrosacea including the use of anti-inflammatories and antibiotics. Thecompositions herein may be used to inhibit, or otherwise treat and/orprevent, rosacea including its erythematotelangiectatic, papulopustular,rosacea and ocular subtypes.

Medical Device, Medical Material, Combination, and PharmaceuticalProducts

The discussion herein also provides medical devices, medical materials,combination, and pharmaceutical products, comprising compositions asdiscussed herein in a medical device, medical material, combinationproduct or pharmaceutically acceptable container. The products can alsoinclude a notice associated with the container, typically in a formprescribed by a governing agency regulating the manufacture, use, orsale of medical devices, medical materials, combination, andpharmaceuticals or biopharmaceuticals, whereby the notice is reflectiveof approval by the agency of the compositions, such as a notice that ahigh-molecular-weight fucan has been approved as an anti-proliferativeagent or anti-inflammatory agent, e.g., for human or veterinaryadministration to treat proliferative diseases or inflammatory diseases(such as, for example, inflammatory arthritis, restenosis, surgicaladhesions, psoriasis and peritonitis). Instructions for the use of thehigh-molecular-weight fucan herein may also be included. Suchinstructions may include information relating to the dosing of a patientand the mode of administration.

The present application is further directed to methods of making thevarious elements of the high-molecular-weight fucan, systems etc.,discussed herein, including making the compositions themselves, as wellas to methods of using the same, including for example treatment of theconditions, diseases, etc., herein.

The present application further comprises medical devices, medicalmaterials, medical combination products, and pharmaceutical products fortreatment of fibrous adhesions, arthritis, psoriasis or other diseasesas desired comprising high-molecular-weight fucans presented herein. Thematerials, etc., can be used in a medicament for treating fibrousadhesions, such as a surgical adhesions, arthritis, psoriasis or otherdiseases as desired. Also provided are methods of manufacturing andusing such medicaments able to reduce symptoms associated with at leastone of fibrous adhesions, arthritis, and psoriasis in a patientincluding a human patient, comprising combining a pharmaceuticallyeffective amount of a fucan such as fucoidan as discussed herein with apharmaceutically acceptable excipient or buffer.

The following Examples provide exemplary discussions of certainembodiments herein but the disclosure and claims are not limitedthereto.

Example 1: Chemical Structural Modification

An exudate-extract was obtained from Laminaria Hyperborea. Theexudate-extract was filtered and small molecules were removed bytangential flow filtration (TFF) over a 100 kDa filter. A sample of theresulting retentate was lyophilized to obtain otherwise unmodifiedsample A. The resulting retentate was brought to 0.25 M NaOH by additionof 10 M NaOH solution and left at room temperature for 16 hours. Theresulting sample was then centrifugally filtered over a 50 kDa filterand the resulting retentate collected and lyophilized to obtainbase-treated sample B. Both unmodified sample A and base-treated sampleB were analyzed by proton nuclear magnetic resonance spectroscopy(¹H-NMR) and the resulting ¹H-NMR spectrum are shown in FIG. 9A.

FIG. 9A demonstrates the chemical structural modification of the fucanaccomplished, the broad peak with a chemical shift about 2.0 ppm that ispresent in the unmodified sample A is not present in the base-treatedsample B.

Unmodified sample A and base-treated/modified sample B were furtheranalyzed by 2D ¹H-¹³C heteronuclear multiple quantum coherence (HMQC).The HMQC spectra, shown in FIG. 9B, were acquired at 70° C. with solventsignal suppression on a 600 MHz spectrometer equipped with 5-mm coldprobe. A high number of scans of the HMQC spectra were acquired in therange from 10-30 ppm in the carbon dimension in 8 increments of 256-512scans each; such scans were combined to create the spectra in FIG. 9B.

The HMQC spectra for unmodified sample A has a cross-peak correspondingto O-acetyl groups, indicated by the signal circled in FIG. 9B. Thiscross-peak is not present in the spectra for base-treated sample B. Thisdemonstrates the removal of acetyl groups from the fucan, and thuschemical structural modification of the fucan in base-treated sample Bby the NaOH treatment.

Example 2: Tangential Flow Filtration

A high-molecular-weight fucan may be obtained by tangential flowfiltration. A broad distribution starting fucan is dissolved indistilled water at 50 mg/mL. In this example, the broad distributionfucan is diafiltered against distilled water over a 100 kDa molecularweight cut-off (MWCO) tangential flow filter (TFF) cassette for 4diavolumes to remove unwanted lower molecular weight components and theretentate of the TFF process is collected comprising thehigh-molecular-weight fucan. The diafiltration may be accomplished withany desired MWCO TFF filter, for example a 50 kDa, 70 kDa, 100 kDa, 300kDa, 500 kDa and 1000 kDa MWCO TFF cassette. The resultinghigh-molecular-weight fucan has a higher average molecular weight thanthe broad molecular weight distribution starting fucan.

Example 3: Sequential Tangential Flow Filtration Segmentation

An input broad molecular weight distribution starting fucoidan having aweight average molecular weight of 365.6 kDa and Polydispersity index(PDI)=3.58 that had been pre-filtered through a 0.22 micron filter wasprovided. A TFF filter cassette of 100 kDa MWCO supplied by Pall of Portof Washington was employed as the higher MWCO TFF cassette and a 50 kDaTFF cassette supplied by Pall of Port of Washington employed as thelower MWCO TFF cassette. The process was repeated for the following TFFcassette pairs: a TFF filter of MWCO 300 kDa supplied by Millipore ofBurlington, Mass. and a TFF filter of MWCO 100 kDa supplied by Pall ofPort of Washington, a TFF filter of MWCO 50 kDa supplied by Pall of Portof Washington and a filter of 30 kDa supplied by Pall of Port ofWashington, a TFF filter of 30 kDa supplied by Pall of Port ofWashington and a TFF filter of 10 kDa supplied by Pall of Port ofWashington. The cassettes were all of the Polyethersulfone (PES) type.

After sequential tangential low filtration as discussed above, thevarious obtained fucans, including high-molecular-weight fucanscomprising high-molecular-weight segments of the starting fucanmolecular weight distribution, were analyzed using gel permeationchromatography (GPC). The results are shown in Table 1 below.

TABLE 1 TFF segmentation of fucoidan GPC % dist. % dist. % dist. PRT PMWWAMW NAMW MW >100 MW >200 MW >500 (Mins) (kDa) (kDa) (kDa) kDa kDa kDaPDI Input 25.51 299.6 365.6 102.2 76.3 57.2 23.1 3.58 MWCO of TFF filterpairs (kDa) 300-100 25.62 278.3 394.2 151.9 83.5 63.3 24.9 2.60 100-50 27.96 59.8 125.1 42.6 37.3 17.4 3.3 2.94 50-30 30.33 12.6 20.6 9.8 1.60.3 0.0 2.11 30-10 34.22 1.0 2.1 1.2 — — — 1.66

Example 4: Cation Augmented Tangential Flow Filtration

A broad molecular weight distribution input starting fucoidancomposition in a starting solution having a weight average molecularweight of 436.4 kDa with a polydispersity index (PDI) of 3.24 that hadbeen pre-filtered through a 0.22 micron filter was provided. Choline, abiocompatible water-soluble quaternary ammonium salt, was selected asthe chemical additive. Choline was added in a 1:2 choline:fucoidan massratio to the pre-filtered starting solution and the resulting mixturestirred until the choline was dissolved. The choline may or may not bindto the sulfate sites on the fucoidan molecules. In a first TFF process,the choline treated-fucoidan solution was then subjected to tangentialflow filtration over a 300 kDa filter cassette to obtain a firstretentate comprising a choline bound high-molecular-weight fucoidan,being a choline-treated retentate. During this first choline-augmentedTFF process, the choline treated-fucoidan solution was diafiltered withfour diavolumes of 1% w/v choline flush solution. The choline-treatedretentate of the first TFF process was collected and subjected to asecond TFF process to replace the choline cations with sodium cations.

The second TFF process, being a decholinating TFF process, compriseddiafiltering the choline-treated retentate of the first TFF process overa 50 kDa filter cassette while treating the retentate with NaCl toreplace the choline cations with sodium cations. In this example, thecholine-treated retentate was diafiltered with 4 volumes of 2 M NaCl toremove the choline additive from the high-molecular-weight fucoidan. Thedecholinated retentate of this second TFF process was then diafilteredwith deionized water until the conductivity of the permeate had droppedto below 5 mS/cm to indicate the removal of excess NaCl. After cationaugmented TFF as discussed above, samples of the various retentates inthe process comprising the high-molecular-weight fucan were analyzedusing gel permeation chromatography (GPC). The results are shown inTable 2 below.

TABLE 2 Cation-augmented TFF segmentation of fucoidan GPC % dist. %dist. % dist. PRT PMW WAMW NAMW MW >100 MW >200 MW >500 (Mins) (kDa)(kDa) (kDa) kDa kDa kDa PDI Input 24.67 436.4 490.7 151.3 82.3 66.1 34.83.24 MWCO of TFF filter retentate (kDa) 300 24.71 423.6 525.5 206.7 88.572.6 37.3 2.54 100 24.10 639.8 740.7 411.0 97.9 90.6 59.1 1.80

Example 5: Centrifugal Precipitation

A starting solution containing 0.5% w/v starting fucoidan compositionthat had been pre-filtered through a 0.22 μm pre-filter was provided. Astep gradient of 20%, 10%, and 5% w/v sucrose in water was created in acentrifuge tube, with 5% layer being the topmost layer and the 20% layerbeing in the bottom of the centrifuge tube. The 0.5% starting solutioncontaining the starting fucoidan composition was then layered on top ofthe 5% w/v sucrose layer. The resulting layer structure is shown in FIG.10. The tube with these four layers was then centrifuged at 190,000gravities (g) for 6 hours. The supernatant solution was decanted and theprecipitate remaining in the centrifuge tube, containing the desiredhigh-molecular-weight fucan, was re-dissolved in water. The re-dissolvedhigh-molecular-weight fucan was then analyzed by gel permeationchromatography (GPC). The results are shown in Table 3 below.

TABLE 3 Centrifugal precipitation of fucoidan using a 5%-10%-20% sucrosebarrier GPC % dist. % dist. % dist. PRT PMW WAMW NAMW MW >100 MW >200MW >500 (Mins) (kDa) (kDa) (kDa) kDa kDa kDa PDI Input 24.57 471.7 590.1200.6 87.4 72.3 40.3 2.94 Re-dissolved 22.95 1472.9 1113.0 492.3 98.291.0 69.1 2.26 fucoidan precipitate

Example 6: Gel Electrophoresis-Extraction

A starting fucoidan composition with a broad molecular weightdistribution was provided. The starting fucoidan composition wasdissolved at 50 mg/mL, pre-filtered through a 0.22 micron filter andloaded onto a 0.5% agarose gel cast from 380 mL of agarose. The loadedgel was submerged in a running buffer of 40 mM tris-acetate 1 mM EDTA,also known as TAE buffer. A voltage of 90 V was applied across thebuffer for 50 minutes with the anode proximate the starting fucoidancomposition well. This allowed the fucoidan to separate by mass tocharge ratio through the gel. For visualization purposes, the gel wasstained with methylene blue, a dye known to stain fucoidan. The agarosegel was then cut in 1 cm wide segments parallel to the well, starting 1cm from the well. The segments of the gel were agitated in distilledwater to extract the fucoidan segments from the gel by shaking themixture.

The electrophoresis-extracted fucoidan segments, potentially comprisinghigh-molecular-weight fucoidans, were analyzed by gel permeationchromatography (GPC). The results are shown in Table 4 below.

TABLE 4 GPC results of the electrophoresis-based separation ofpre-filtered starting fucoidan across agarose gel with TAE buffer. GPC %dist. % dist. % dist. PRT PMW WAMW NAMW MW >100 MW >200 MW >500 (Mins)(kDa) (kDa) (kDa) kDa kDa kDa PDI Input 24.67 462.6 581.3 170.9 86.773.0 40.8 3.40 Distance of gel segment from well 1-2 cm 25.07 349.4619.9 81.33 71.3 55.9 30.7 7.62 2-3 cm 25.16 327.8 362.0 118.2 76.2 56.723.9 3.06 3-4 cm 25.47 263.6 288.4 103.7 70.9 48.8 16.7 2.78 4-5 cm25.58 242.7 279.1 66.0 62.8 42.2 14.9 4.23

Example 7: Membrane Dialysis

A 5% w/v starting solution containing a starting fucoidan compositionthat had been pre-filtered through a 0.22 micron filter was provided.The starting solution was placed in a cellulose acetate dialysis tubingof nominal molecular weight cutoff 300 kDa. The dialysis tubing wassealed and placed in a container with 20 liters of deionized water. Thedeionized water was replaced with fresh deionized water every 12 hoursto ensure continuous diffusion across the membrane pores. The dialysisprocess was allowed to continue for about 5 days.

The pre-filtered starting fucoidan composition and the post-dialysis,high-molecular-weight fucoidan in the dialysis tube were both analyzedusing gel permeation chromatography (GPC). The results are shown inTable 5 below.

TABLE 5 GPC results of the dialysis of fucoidan against deionized wateracross a 300 kDa membrane. GPC % dist. % dist. % dist. PRT PMW WAMW NAMWMW >100 MW >200 MW >500 (Mins) (kDa) (kDa) (kDa) kDa kDa kDa PDI Input24.57 471.7 590.1 200.6 87.4 72.4 40.3 2.94 Dialyzed 24.29 599.1 777.0374.5 96.8 88.6 57.0 2.07 Fucoidan

Example 8: Selective Precipitation

A starting fucoidan composition that had been pre-filtered through a0.22 μm pre-filter and desalted via diafiltering with deionized waterover a 100 kDa TFF cassette to remove unwanted low molecular weightsalts that may interfere with the precipitation process was provided. Aseries of identical starting solutions of the prefiltered and desaltedstarting fucoidan in distilled water were prepared. The solventcompositions were brought up to different pre-determined concentrationsof ethanol. This prepared the different solvent environments for theprecipitation of the fucoidan from the solution compositions identifiedin Table 6 below. A minimal amount of an ionic agent in the form of NaClwas added to each solution composition to initiate the precipitation offucoidan from the solution. The mixes of precipitate and solutioncomposition were centrifuged at 2300 gravities for 10 minutes. Theliquid supernatant was decanted in each case and the solid fucoidancollected.

The solid fucoidan were re-dissolved in distilled water and analyzed bygel permeation chromatography. The results are shown in Table 6 below.

TABLE 6 Selective precipitation of fucoidan using ethanol asprecipitating solvent % Ethanol in GPC % dist. % dist. % dist. thefucoidan PRT PMW WAMW NAMW MW >100 MW >200 MW >500 solution (Mins) (kDa)(kDa) (kDa) kDa kDa kDa PDI 40 24.85 394.1 447.0 133.5 76.6 62.5 31.33.35 50 25.96 182.1 347.9 149.2 80.8 53.9 20.3 2.33 60 25.43 263.1 335.5119.1 73.9 51.9 20.0 2.82 70 24.91 376.1 382.6 117.2 75.7 56.8 25.4 3.26

Example 9: Anionic Adsorption

A starting solution containing about 500 mg of a broad molecular weightdistribution desalted starting fucoidan was recirculated on about 14 mLof DEAE-Sepharose® resin for about 16 hours to bind the low molecularweight fucoidan to the active sites on the resin. After about 16 hoursthe recirculating solution was collected. This separated thehigh-molecular-weight fucoidan from the low molecular weight fucoidanthat had bonded to the resin. 10% w/v NaCl was then recirculated on theresin for 4 hours to displace the low molecular weight fucoidan from theresin. The fucoidan rich-salt solution was then collected and desaltedover a 5 kDa centrifugal filter to separate the collected low molecularweight fucoidan from the unwanted salt. GPC was performed on thedesalted starting fucoidan, the high-molecular-weight fucoidan notadsorbed during the ion exchange process, and the low molecular weightfucoidan extracted from the resin. The results are shown in Table 7below.

TABLE 7 Anion exchange segmentation of fucoidan: DEAE-Sepharose as resinGPC % dist. % dist. % dist. PRT PMW WAMW NAMW MW >100 MW >200 MW >500(Mins) (kDa) (kDa) (kDa) kDa kDa kDa PDI Input 24.57 462.4 576.6 198.087.1 71.9 39.6 2.91 Fucoidan 24.20 601.3 844.5 391.4 96.8 90.9 60.5 2.16not adsorbed Fucoidan 25.82 193.6 245.8 119.2 73.0 43.8 10.7 2.06adsorbed

Example 10: Anionic Adsorption

A starting solution containing about 1 g of a broad molecular weightdistribution desalted fucoidan was mixed with about 10 g Amberlyst® A26resin for about 16 hours to bind the low molecular weight fucoidan tothe active sites on the resin. The solution containing thehigh-molecular-weight fucoidan was subsequently separated from the resinby decanting. 20% w/v NaCl was then mixed with the resin for about 4hours to displace the low molecular weight fucoidan from the resin. Thefucoidan rich salt solution was then separated from the resin anddesalted over a 5 kDa centrifugal filter to separate the collected lowmolecular weight fucoidan from the unwanted salt. GPC was performed onthe desalted starting fucoidan, the high-molecular-weight fucoidan notadsorbed during the ion exchange process, and the low molecular weightfucoidan extracted from the resin. The results are shown in Table 8below.

TABLE 8 Anion exchange separation of fucoidan: Amberlyst ™ A26 OH GPC %dist. % dist. % dist. PRT PMW WAMW NAMW MW >100 MW >200 MW >500 (Mins)(kDa) (kDa) (kDa) kDa kDa kDa PDI Input 25.02 517.7 536.9 148.2 82.767.7 38.1 3.62 Fucoidan 24.73 625.8 867.4 463.1 98.7 93.0 62.6 1.87 notadsorbed Fucoidan 27.30 112.4 172.3 86.4 58.0 25.4 4.7 2.00 adsorbed

Example 11: Anionic Adsorption

A starting solution containing about 1 g of a broad molecular weightdistribution desalted fucoidan was mixed with about 10 g of threedifferent resins, being Amberlyst® A26 OH⁻, Ambersep® 900 OH⁻, andLewatit® VPOC 1065 in three separate containers. The solution-resinmixtures were incubated for about 16 h to bind the low molecular weightfucoidan to the active sites on the resin. The solution containing thehigh-molecular-weight fucoidan was subsequently separated from the resinby decanting. The pores of the Amberlyst® and Ambersep® product hadquaternary amine groups while the pores of the Lewatit product hadprimary benzylamine groups. The first two products were strongly basicanion exchange resins, while the third was a weakly basic anion exchangeresin. The fucoidan not adsorbed during the ion-exchange process werethen analyzed by GPC. The results are shown in Table 9 below.

TABLE 9 Anion exchange separation of fucoidan: comparison of fucoidansprepared by recirculation on 3 resins. GPC % dist. % dist. % dist. PRTPMW WAMW NAMW MW >100 MW >200 MW >500 (Mins) (kDa) (kDa) (kDa) kDa kDakDa PDI Input 24.66 461.9 521.5 151.8 83.0 67.3 36.5 3.44 Resin usedAmbersep ® 24.51 513.0 609.1 323.6 96.1 85.5 47.3 1.88 900 OH⁻Amberlyst ® 24.58 489.8 591.0 309.5 95.6 83.5 45.1 1.91 A26 OH⁻Lewatit ® 24.54 501.2 585.6 219.6 89.5 75.3 42.4 2.67 VPOC 1065

Example 12: Anionic Adsorption

A starting solution containing about 1 g of a broad molecular weightdistribution desalted fucoidan was mixed with about 10 g Ambersep® 900OH⁻ for up to 53 hours. The fucoidan in the mixture not adsorbed duringthe ion-exchange process were then analyzed by GPC at various timepoints during the anion adsorption process. The results are shown inTable 10 below.

TABLE 10 Anion exchange separation of fucoidan: comparison of anionexchange times GPC % dist. % dist. % dist. PRT PMW WAMW NAMW MW >100MW >200 MW >500 (Mins) (kDa) (kDa) (kDa) kDa kDa kDa PDI Input 26.71624.2 955.9 339.9 95.2 84.6 56.3 2.81 Ion exchange time (hours) 1 26.66642.4 1049.2 386.0 96.6 87.3 59.5 2.72 4 26.42 756.4 1151.7 470.9 98.291.7 65.5 2.45 24 26.33 801.9 1205.2 589.9 99.5 95.9 72.1 2.04 53 26.25843.6 1257.9 656.9 99.8 97.5 75.6 1.91

Example 13: Anionic Adsorption

A starting solution containing about 1 g of the broad molecular weightdistribution desalted fucoidan was mixed with various amounts ofAmbersep® 900 OH⁻ for about 16 hours to bind the low molecular weightfucoidan to the active sites on the resin. The solution containing thehigh-molecular-weight fucoidan was subsequently separated from the resinby decanting. The fucoidan not adsorbed during the ion-exchange processwere then analyzed by GPC. The results are shown in Table 11 below.

TABLE 11 Anion exchange separation of fucoidan: comparison of differentfucoidan to resin ratios GPC % dist % dist. % dist. PRT PMW WAMW NAMWMW >100 MW >200 MW >500 (Mins) (kDa) (kDa) (kDa) kDa kDa kDa PDI Input24.66 442.4 498.5 146.3 82.4 66.1 34.8 3.41 Mass ratio of Fucoidan:resin1:1 24.51 491.1 548.1 203.2 87.6 72.0 39.0 2.70 1:5 24.48 500.9 633.9306.6 95.3 82.9 46.6 2.07  1:10 24.41 523.8 688.1 376.4 98.0 88.8 51.91.83

Example 14: Preparative Gel Permeation Chromatography

A starting fucoidan composition with a broad molecular weightdistribution is provided. The starting fucoidan composition is dissolvedat 10 mg/mL in 60 mL 0.1 M sodium nitrate. 20 mL of the startingsolution containing the starting fucoidan composition is pumped at 40mL/min through each of a 50 mm inner diameter, 250 mm length columncontaining Sepax® SRT-10/10C SEC-1000, Agilent® PL Aquagel®-OH MIXED-Hand TSKGel® G4000SW respectively, all of which contain modifiedsilica-hydrophilic bonded phase gel media. Elution is carried out at thesame flow rate using 0.1 M sodium nitrate. After 5 minutes of elution,40 mL aliquots are collected until a total of 1000 mL, or 25 aliquots,have been collected. The molecular weight distribution of a sample ofeach aliquot is measured by analytical GPC. Aliquots containing weightaverage molecular weights between 200 kDa and 600 kDa are pooled.Aliquots containing weight average molecular weights between 600 kDa and1000 kDa are pooled. Aliquots containing weight average molecularweights between 1000 kDa and 1400 kDa are pooled. Aliquots containingweight average molecular weights between 1400 kDa and 1800 kDa arepooled. The rest of the aliquots are discarded. Each pooled preparativeGPC aliquot composition contains a desired high-molecular-weight fucan.

Example 15: Preparative Gel Permeation Chromatography

A starting fucoidan composition with a broad molecular weightdistribution is provided. The starting fucoidan composition is dissolvedat 10 mg/mL in 60 mL 0.1M sodium nitrate. 20 mL of the starting solutioncontaining the starting fucoidan composition is pumped at 40 mL/minthrough each of a 50 mm inner diameter, 250 mm length column containingWaters® HSPgel AQ MB-H, PSS® Suprema® Combination Ultrahigh and TSKGel®GMPWXL respectively, all of which contain hydroxylatedpolymethacrylate-based gel media. Elution is carried out at the sameflow rate using 0.1M sodium nitrate. After 5 minutes of elution, 40 mLaliquots are collected until a total of 1000 mL, or 25 aliquots, havebeen collected. The molecular weight distribution of a sample of eachaliquot is measured by analytical GPC. Aliquots containing at least 90%of their molecular weight distribution above 100 kDa, at least 80% oftheir molecular weight distribution above 200 kDa and/or at least 50% oftheir molecular weight distribution above 500 kDa are pooled. The restof the aliquots are discarded. Each pooled preparative GPC aliquotcomposition contains a desired high-molecular-weight fucan.

Example 16: Preparation of Low and High-Molecular-Weight Fucans

The methods discussed herein may be used, combined, modified andpermuted in any manner to obtain high-molecular-weight fucans.

Twenty fucans, some with high and some with low molecular weights, wereprepared from feedstock/starting fucan compositions having broadmolecular weight distributions to evaluate the efficacy of high and lowmolecular weight fucans in medical and surgical applications. The twentyfucans are hereafter referred to as fucan 1 to fucan 20. Fucan 1 tofucan 5 were light brown solids. Fucan 6, fucan 8 to fucan 15 and fucan17 were white solids. The preparation of low-molecular weight fucans,being fucan 1 to fucan 6, involved numerous different methodologies.Fucan 3 was extracted from brown seaweed and found to be a low-molecularweight fucan. Fucan 2 was obtained from FMC BioPolymer® and found to bea low-molecular weight fucan. Fucan 1 and fucan 5 were obtained bymethods discussed in example 3, using MWCO TFF filters under 100 kDa.Fucan 4 was obtained by methods discussed in example 10. Fucan 6 wasobtained by chemical degradation of a high-molecular-weight fucan withhydrogen peroxide.

The preparation of high-molecular-weight fucans, being fucan 7 to fucan20, involved numerous different methodologies including treatment withsodium hydroxide, and in some cases other bases as well. The preparationof fucan 7, fucan 8, fucan 11, fucan 12 to fucan 17 and fucan 20involved a combination of the method discussed in example 12 andtangential flow filtration against a low ionic strength solution. Thepreparation of fucan 10 involved a combination of cationic augmentedtangential flow filtration and sequential tangential flow filtrationmethods discussed above. Fucan 9, fucan 18 and fucan 19 were extractedfrom brown seaweed, further processed by tangential flow filtrationagainst a low ionic strength solution and found to behigh-molecular-weight fucans.

Example 17: Molecular Weight Determination of Crude Fucans Used to MakeFucan 7 to Fucan

Gel permeation chromatography was used to evaluate the molecular weightdistributions of crude fucans used to make fucans 7 to 14. Crude fucan 1refers to the crude fucan used to make fucan 7 and fucan 8. Crude fucan2 refers to the crude fucan used to make fucan 9, fucan 10, fucan 11 andfucan 13. Crude fucan 3 refers to the crude fucan used to make fucan 12.Crude fucan 4 refers to the crude fucan used to make fucan 14. Theresults of such analyses are shown in Table 12.

Results in the tables below contain abbreviations used for certaincharacteristics of a molecular weight distribution. Gel permeationchromatography is denoted by GPC, peak molecular weight is denoted byPMW, weight average molecular weight is denoted by WAMW, number averagemolecular weight is denoted by NAMW, percentage distribution is denotedby % dist., molecular weight is denoted by MW and polydispersity indexis denoted by PDI.

TABLE 12 % % % % % % PMW WAMW NAMW dist. <10 dist. <20 dist. <50dist. >100 dist. >200 dist. >500 (kDa) (kDa) (kDa) kDa kDa kDa kDa kDakDa PDI Crude 92.0 259.3 22.1 8.3 14.7 30.4 52.3 34.7 14.5 11.7 fucan 1Crude 512.3 535.3 128.5 0.5 2.1 8.9 80.4 65.1 36.6 4.2 fucan 2 Crude594.6 493.4 4.4 22.0 27.3 35.7 57.4 49.3 31.3 113.3 fucan 3 Crude 662.5790.6 245.4 0.1 0.5 3.4 90.9 80.2 52.0 3.2 fucan 4

Example 18: Molecular Weight Determination of Low andHigh-Molecular-Weight Fucans

Gel permeation chromatography was used to evaluate the molecular weightdistributions obtained for fucans 1 to 20.

Table 13 and Table 14 list the molecular weight distribution profilesobtained for twenty fucans. Table 14 provides molecular weightdistribution profiles for the same twenty fucans shown in Table 13,providing the molecular weight distribution profiles in a differentmanner that shown in Table 13, providing thereby two differentperspectives on the molecular weight distribution of the various fucans.As can be seen from the results, a broad range of different molecularweight distributions in fucans has been accomplished. Fucans with aweight average molecular weight between 28 kDa and 8250 kDa have beenobtained with a plurality of distribution profiles.

Results in the tables below contain abbreviations used for certaincharacteristics of a molecular weight distribution. Gel permeationchromatography is denoted by GPC, peak molecular weight is denoted byPMW, weight average molecular weight is denoted by WAMW, number averagemolecular weight is denoted by NAMW, percentage distribution is denotedby % dist., molecular weight is denoted by MW and polydispersity indexis denoted by PDI.

TABLE 13 A first perspective on the molecular weight distribution of 20fucans % % % % % % PMW WAMW NAMW dist. <10 dist. <20 dist. <50dist. >100 dist. >200 dist. >500 (kDa) (kDa) (kDa) kDa kDa kDa kDa kDakDa PDI Fucan 1 17.5 28.3 14.2 16.6 51.7 87.9 3.0 0.6 0.0 1.99 Fucan 221.0 72.4 9.9 26.5 44.0 67.3 18.4 9.1 2.3 7.29 Fucan 3 70.4 105.9 52.40.6 5.8 33.1 35.3 12.1 1.3 2.02 Fucan 4 107.1 136.1 79.9 0.1 1.5 15.453.4 19.8 1.1 1.70 Fucan 5 80.2 171.9 60.4 0.8 5.3 26.5 47.9 25.4 6.62.84 Fucan 6 195.1 192.1 87.4 0.4 2.3 14.0 64.4 35.8 5.5 2.20 Fucan 7242.5 366.5 137.2 0.0 0.5 7.0 77.7 54.6 21.9 2.67 Fucan 8 307.1 395.8170.2 0.0 0.2 4.0 83.8 62.2 25.4 2.33 Fucan 9 459.3 514.0 198.5 0.1 0.43.4 87.8 71.4 37.2 2.62 Fucan 10 390.2 497.9 228.9 0.0 0.0 1.7 90.4 73.335.1 2.17 Fucan 11 457.3 592.8 300.9 0.0 0.0 0.7 95.4 82.9 43.8 1.97Fucan 12 535.8 760.1 350.6 0.0 0.1 0.9 96.5 88.3 54.3 2.17 Fucan 13612.3 857.0 448.7 0.0 0.0 0.2 98.6 92.4 61.4 1.91 Fucan 14 393.1 930.1296.6 0.0 0.0 1.1 93.6 81.1 43.6 3.14 Fucan 15 409.4 772.0 291.8 0.0 0.01.1 94.0 81.5 43.6 2.65 Fucan 16 743.0 1618.0 387.5 0.0 0.1 1.4 92.986.6 68.2 4.18 Fucan 17 686.2 1876.7 524.9 0.0 0.0 0.3 98.4 93.0 69.93.58 Fucan 18 6238.6 3957.4 519.7 0.0 0.1 1.7 82.3 78.8 71.4 7.61 Fucan19 4315.2 5336.8 2009.5 0.0 0.0 0.0 93.7 93.3 90.1 2.66 Fucan 20 6170.28101.9 846.3 0.0 0.0 0.3 94.7 91.1 83.6 9.57

TABLE 14 A second perspective on the molecular weight distribution of 20fucans % dist. <5 % dist. between % dist. between % dist. between %dist. >1600 kDa 5-60 kDa 60-200 kDa 200-1600 kDa kDa Fucan 1 3.5 87.98.1 0.6 0.0 Fucan 2 13.0 58.6 19.4 9.0 0.0 Fucan 3 0.0 41.3 46.6 12.10.0 Fucan 4 0.0 20.9 59.1 20.1 0.0 Fucan 5 0.1 32.8 41.7 25.0 0.4 Fucan6 0.0 18.5 45.6 35.8 0.0 Fucan 7 0.0 10.0 35.4 52.3 2.4 Fucan 8 0.0 6.231.5 60.0 2.3 Fucan 9 0.0 5.0 23.6 67.4 4.0 Fucan 10 0.0 3.0 23.7 69.83.5 Fucan 11 0.0 1.3 15.8 78.0 4.9 Fucan 12 0.0 1.3 10.5 78.9 9.4 Fucan13 0.0 0.3 7.2 80.4 12.0 Fucan 14 0.0 1.8 16.3 68.7 13.1 Fucan 15 0.01.7 16.1 72.4 9.8 Fucan 16 0.0 2.1 9.4 60.9 37.6 Fucan 17 0.0 0.5 6.562.4 30.6 Fucan 18 0.0 2.3 5.7 35.2 56.8 Fucan 19 0.0 0.0 0.3 24.9 74.8Fucan 20 0.0 0.6 5.2 28.7 65.5

Example 19: Sulfate, Total Carbohydrate and Monosaccharide Content ofHigh-Molecular-Weight Fucans

High-molecular-weight fucans fucan 7 to fucan 18 and fucan 20 weredissolved in deionized water, hydrolyzed under acidic conditions andanalyzed by inductively coupled plasma mass spectrometry (ICP-MS) for %w/w total sulfur content, performed by ALS Environmental laboratories inBurnaby, British Columbia. Sulfur content was converted to sulfatecontent by multiplying the sulfur content by the molar ratio of sulfateto sulfur to obtain % w/w sulfate content of the fucan. The sulfatecontents of fucan 7 to 18 and fucan 20 are shown in table 15 below.

TABLE 15 Sulfate content of fucan 7 to fucan 18 and fucan 20 Sulfatecontent (% w/w) Fucan 7 23.93 Fucan 8 40.95 Fucan 9 40.32 Fucan 10 33.15Fucan 11 44.87 Fucan 12 41.02 Fucan 13 36.18 Fucan 14 40.45 Fucan 1539.79 Fucan 16 14.39 Fucan 17 51.30 Fucan 18 21.11 Fucan 20 25.60

High-molecular-weight fucans fucan 7, fucan 11, fucan 16, fucan 18 andfucan 20 were analyzed for total carbohydrate and monosaccharidecomposition by gas spectrometry-mass spectroscopy (GC-MS) performed bythe complex carbohydrate research center at the University of Georgia.The high-molecular-weight fucans were derivatized by acidic methanolysisto produce O-trimethylsilyl (O-TMS) derivatives. After derivatization,the fucans are analyzed on an Agilent 7890A gas chromatography systeminterfaced to an Agilent 5975C mass spectrometry detector using aSupelco Equity-1 fused silica capillary column (30 m, 0.25 mm innerdiameter). The results for the total carbohydrate content and themonosaccharide composition of the high-molecular-weight fucans are shownin table 16 below. Carbohydrate in the table below is abbreviated“carb.”.

TABLE 16 Total carbohydrate and monosaccharide composition of fivefucans Total carb. Fucose Galactose Xylose Mannose Rhamnose content (%w/w of (% w/w of (% w/w of (% w/w of (% w/w of (% w/w of the total carb.total carb. total carb. total carb. total carb. the fucan) content)content) content) content) content) Fucan 7 32.7 44.4 52.9 0.5 0.4 0.3Fucan 11 59.5 91.9 8.1 0.0 0.0 0.0 Fucan 16 25.9 48.3 9.9 15.5 5.9 0.3Fucan 18 41.2 92.0 4.7 2.1 0.4 0.2 Fucan 20 30.1 84.7 10.6 3.3 0.9 0.0

Example 20: Rat Epidural Adhesion Treatment

Fucoidan solutions using the twenty fucans identified in the example 18were prepared in Lactated Ringers Injection USP (LRS). Fucan 1 to fucan16, fucan 18 and fucan 20 were prepared at 100 mg/mL in LRS. Fucan 19was prepared at 50 mg/mL in LRS. Fucan 17 was prepared at 500 mg/mL inLRS. Laminectomy surgery was performed on Sprague Dawley rats, theaverage weights of the rats and the dose in milligram per kilogram shownin table 17 below. A line block along the lumbar spine was created withbupivacaine solution. The back of the rat was cleaned and then coveredwith sterile drapes. The lumbar fascia was opened through a midline skinincision, lumbosacral fascia was incised and the paralumbar muscles wasdissected to expose the underlying vertebral laminae. Bone at the centreof the vertebrae was removed. Throughout the procedure, haemostasis wasmaintained by irrigation with Lactated Ringer's Injection USP (LRS) andpressure with cotton swabs. The exposed dura was treated directly with15 microlitres of LRS (control) or fucoidan solution. The muscle andskin layers were closed with sutures and the rats were allowed torecover for one week before sacrifice for adhesion quantification. Thepresence and size of adhesions on the dura were noted. The dimensions ofthe adhesions and the exposed dura were recorded and used to calculatean adhesion coverage percentage, being the adhesion area as a percentageof the total exposed dura area.Adhesion coverage (%)=100×epidural adhesion area÷total exposed duraarea  Equation 1:

The control group receiving LRS was determined to have a 65% adhesioncoverage using equation 1. The adhesion coverage for the twenty fucansdisclosed in Table 13 to Table 16 are shown in Table 17 below as thereduction in adhesion coverage relative to the control group. A negativevalue denoted where an increase in adhesion coverage was seen relativeto the control group.

TABLE 17 Reduction in rat epidural adhesion relative to control LRSusing 20 different fucans Average Dose per % Reduction in Rat animalNumber epidural Weight Dose weight of rats adhesion coverage (kg) (mg)(mg/kg) scored vs. control Fucan 1 0.41 1.5 3.7 4 −40% (i.e., 40%increase in fibrous adhesions compared to control) Fucan 2 0.59 1.5 2.53  9% Fucan 3 0.39 1.5 3.8 4 −10% Fucan 4 0.65 1.5 2.3 4  83% Fucan 50.53 1.5 2.9 4 46% (i.e., 46% decrease in fibrous adhesions compared tocontrol) Fucan 6 0.46 1.5 3.3 4  44% Fucan 7 0.47 1.5 3.2 3 100% Fucan 80.36 1.5 4.2 3 100% Fucan 9 0.39 1.5 3.8 2 100% Fucan 10 0.40 1.5 3.8 4100% Fucan 11 0.58 1.5 2.6 2 100% Fucan 12 0.44 1.5 3.4 2 100% Fucan 130.64 1.5 2.3 3 100% Fucan 14 0.37 1.5 4.0 4 100% Fucan 15 0.50 1.5 3.0 3100% Fucan 16 0.45 1.5 3.3 3 100% Fucan 17 0.59 7.5 12.8 3 100% Fucan 180.59 1.5 2.5 2 100% Fucan 19 0.39 0.8 1.9 3 100% Fucan 20 0.56 1.5 2.7 2100%

As may be seen by comparing the results of Table 17 with the molecularweight of the fucans given in Tables 13 and Table 14, fucans with aweight average molecular weight over 130 kDa and containing about 60% ormore of their molecular weight distribution above 100 kDa show greaterefficacy in the inhibition, prevention, removal, reduction, or othertreatment of rat epidural adhesions than fucans with weight averagemolecular weight below 100 kDa containing about 60% or less of theirmolecular weight distribution above 100 kDa at the same dose. There isalso a further indication that fucans with weight average molecularweight above 300 kDa, containing about 70% or more of their molecularweight distribution above 100 kDa show even greater efficacy in theinhibition, prevention, removal, reduction, or other treatment of ratepidural adhesions at the same dose.

Example 21: Rabbit Uterine Horn Adhesion Treatment with Fucan 1 andFucan 10

Uterine horn surgery was performed on both uterine horns in each rabbit.Prior to surgery, the rabbits were weighed and then prepared for surgeryby premedication with ketamine and xylazine.

Fucoidan solution was prepared at 0.07 mg/mL in Lactated RingersInjection USP, sterilizing by filtration. All instruments were sterile,and a sterile field was maintained throughout the surgeries. The abdomenwas cleaned and entered via a midline abdominal incision. The uterinehorns were located, exteriorized and scraped to induce damage. Theabdominal wall near the scraped uterine horns was also scraped. Thedamaged uterine horns and abdominal wall were placed next to each otherand stabilized with sutures. 15 mL/kg fucoidan solution per rabbitweight was applied to the abdominal cavity before the incision wasclosed. Adhesion was evaluated two weeks after the surgery. Length ofthe uterine horn adhesion was measured with a ruler. The uterine hornadhesion coverage percentage, being the length of the adhesion as apercentage of the total damaged uterine horn length was calculated as:Adhesion coverage (%)=100×uterine horn adhesion length÷total damageduterine horn length  Equation 2:

The same surgical method was applied to 3 New Zealand White rabbits,receiving 15 mL/kg of control Lactated Ringer's Injection USP (LRS)instead of fucoidan solution.

The control group receiving LRS was determined to have a 41% adhesioncoverage using equation 2. Table 18 shows the results obtained using themethod discussed above for fucans fucan 1 and fucan 10, beingrepresentative examples of respectively a fucan with the majority of itsmolecular weight distribution below 100 kDa and even below 50 kDa and afucan with the majority of its molecular weight distribution above 100kDa and even above 200 kDa. The results in the table below are shown asthe reduction in adhesion coverage relative to the control group.

TABLE 18 Reduction in rabbit uterine horn adhesion using two differentfucans relative to control LRS % Reduction in Dose per Number of uterinehorn animal weight Uterine adhesion coverage (mg/kg) Horns vs. controlFucan 1 - low 1 6 21% (i.e., 21% decrease molecular weight in fibrousadhesions compared to control) Fucan 10 - high- 1 8 100%molecular-weight

As may be seen from the results in Table 18, fucans having the majorityof their distribution above 100 kDa, or even above 200 kDa, have ahigher efficacy in the inhibition, prevention, removal, reduction, orother treatment of rabbit uterine horn adhesion as compared with fucanshaving a majority of their distribution under 100 kDa or even under 50kDa at the same dose.

Example 22: Rabbit Uterine Horn Adhesion with Fucan 17

To determine the efficacy of the high-molecular-weight fucan 17 ininhibiting surgical adhesions, the following double uterine horn (DUH)surgeries were performed on both horns of a total of three New ZealandWhite rabbits. Prior to surgery, the rabbits were weighed and thenprepared for surgery by premedication with ketamine and xylazine.

Fucoidan solution was prepared at 5 mg/mL in Lactated Ringers InjectionUSP (LRS), sterilizing by filtration. All instruments were sterile, anda sterile field was maintained throughout the surgeries. The abdomen wascleaned and entered via a midline abdominal incision. The uterine hornswere located, exteriorized and scraped to induce damage. The abdominalwall near the scraped uterine horns was also scraped. The damageduterine horns and abdominal wall were placed next to each other andstabilized with sutures. The top third and the bottom third of themuscle incision was closed and 5 mL/kg fucoidan solution per rabbitweight was applied to the abdominal cavity. The muscle incision wastemporarily closed and the fucoidan solution left in the abdominalcavity for 30 minutes. The muscle incision was reopened and theabdominal cavity was flushed with 10 mL/kg LRS. The majority of thefluid in the abdominal cavity was suctioned out before the incision wasclosed. Adhesion formation was evaluated two weeks after the surgery.Length of the uterine horn adhesion was measured with a ruler. Theuterine horn adhesion coverage percentage, being the length of theadhesion as a percentage of the total damaged uterine horn length wascalculated using equation 2.

Table 19 shows the results obtained using the method discussed above forfucan 17, being a representative example of a high-molecular-weightfucan. The results in the table below are shown as the mean adhesionlength across the 6 uterine horns scored.

Table 19 provides the results of treating six uterine horns with fucan17.

TABLE 19 Adhesion length using fucan 17 Number of Mean % Dose Uterineadhesion (mg/kg) Horns length Fucan 17 25 6 0% (i.e., no adhesions werefound)

As may be seen from the results of Table 19, high-molecular-weightfucans may be used to successfully inhibit, prevent, remove, reduce, orotherwise treat post-surgical uterine horn adhesions.

Example 23: Uterine Horn Fibrous Adhesion Treated with aHigh-Molecular-Weight Fucan Composition

To determine the efficacy of a high-molecular-weight fucan compositioncomprising a number average molecular weight of about 228 kDa, a weightaverage molecular weight of about 1210 kDa, a peak molecular weight ofabout 575 kDa and having a molecular weight distribution wherein about89% of the distribution is above 100 kDa and wherein about 30% of thedistribution is above 1000 kDa, in inhibiting surgical adhesions, thefollowing double uterine horn (DUH) surgeries were performed on bothhorns of a total of twenty New Zealand White rabbits. Prior to surgery,the rabbits were weighed and then prepared for surgery by premedicationwith midazolam and dexmeditomidine.

Fucoidan solution was prepared at each concentration of 0.02 mg/mL, 0.1mg/mL, 0.5 mg/mL, or 2.5 mg/mL in Lactated Ringers Injection USP (LRS),sterilizing by filtration. All instruments were sterile, and a sterilefield was maintained throughout the surgeries. The abdomen was cleanedand entered via a midline abdominal incision. The uterine horns werelocated, exteriorized and scraped to induce damage. The abdominal wallnear the scraped uterine horns was also scraped. The damaged uterinehorns and abdominal wall were placed next to each other and stabilizedwith sutures. About 2 mL/kg fucoidan solution per rabbit weight wasapplied to the abdominal cavity before the incision was closed. Adhesionwas evaluated two weeks after the surgery. Five rabbits were treated andevaluated for each fucoidan concentration prepared. Length of theuterine horn adhesion was measured with a ruler. The uterine hornadhesion length was calculated using equation 2.

The same surgical method was applied to 5 additional New Zealand Whiterabbits for control, each receiving about 2 mL/kg of control LactatedRinger's Injection USP (LRS) instead of fucoidan solution. The controlgroup receiving LRS was determined to have a 100% adhesion coverageusing equation 2. Table 20 shows the results obtained using the methoddiscussed above for the high-molecular-weight fucan composition atdifferent concentrations and dosages (in total forty uterine horns weretreated, 10 each for each concentration of the high-molecular-weightfucan composition); the results are shown as the reduction in adhesioncoverage relative to the control group.

TABLE 20 Decrease in rabbit uterine horn adhesion using ahigh-molecular- weight fucan composition relative to control LRS %Reduction in Number of uterine horn Concentration Dose Uterine adhesioncoverage (mg/mL) (mg/kg) Horns vs. control 0.02 0.04 10 10% (i.e., 10%decrease in fibrous adhesions compared to control) 0.1 0.2 10 30% (i.e.,30% decrease in fibrous adhesions compared to control) 0.5 1 10 71%(i.e., 71% decrease in fibrous adhesions compared to control) 2.5 5 1095% (i.e., 95% decrease in fibrous adhesions compared to control)

As can be seen from the results of Table 20, high-molecular-weight fucancompositions can be used to successfully inhibit, prevent, remove,reduce, or otherwise treat post-surgical uterine horn adhesions.

REFERENCE NUMERAL LIST

-   100 Molecular weight based segmentation system (higher-to-lower)-   100′ Molecular weight based segmentation system (lower-to-higher)-   100″ Cation-augmented TFF system (CATS)-   102 Input supply line-   104 Pre-filter-   106 Lower MWCO subsystem retentate-line valve-   106′ Lower MWCO subsystem output valve-   108 lower MWCO subsystem retentate output line-   110 Higher molecular weight cut-off TFF filter-   111 Higher MWCO subsystem retentate output line-   112 Higher MWCO TFF filter supply line-   113 Higher-to-lower MWCO inter-subsystem valve-   114 Higher MWCO subsystem pump-   115 Higher MWCO subsystem solvent supply line-   116 Higher MWCO subsystem fucan container-   117 Higher MWCO subsystem solvent container-   118 Higher MWCO subsystem retentate return line-   119 Higher MWCO subsystem permeate output line-   120 Lower molecular weight cut-off TFF filter-   121 Lower MWCO subsystem retentate output line-   122 Lower MWCO TFF filter supply line-   123 Lower-to-higher MWCO inter-subsystem valve-   124 Lower MWCO subsystem pump-   125 Lower MWCO subsystem solvent supply line-   126 Lower MWCO subsystem fucan container-   127 Lower MWCO subsystem solvent container-   128 Lower MWCO subsystem retentate return line-   129 Lower MWCO subsystem permeate output line-   130 Higher MWCO TFF subsystem-   130′ Higher MWCO TFF subsystem (FIG. 3)-   135 Cationic additive flush solution supply line-   136 Cationic additive flush solution valve-   137 Cationic additive flush solution container-   140 Lower MWCO subsystem-   140′ Lower MWCO TFF subsystem (FIG. 3)-   142 Sodium salt solution container-   143 Low conductivity diafiltration solution container-   144 Sodium salt solution control valve-   145 Low conductivity diafiltration solution valve-   146 Sodium salt solution supply line-   147 Low conductivity diafiltration solution supply line-   150 Higher MWCO TFF filter-   160 Lower MWCO TFF filter-   600 Centrifugal precipitation system for obtaining a    high-molecular-weight fucan from a starting fucan composition-   600′ Centrifugal precipitation system for obtaining a    high-molecular-weight fucan from a starting fucan composition-   610 Centrifuge container-   620 Gradated permeable barrier-   620′ Permeable barrier-   620 a Barrier segment (first—highest density)-   620 b Barrier segment (second—intermediate density)-   620 c Barrier segment (third—lowest density)-   620 c′ Single barrier segment-   622 First-bottom gradated permeable barrier material end-   622′ First-bottom permeable barrier material end-   624 Second-top gradated permeable barrier material end-   624′ Second-top permeable barrier material end-   630 First-bottom end of centrifuge container 610-   640 Second-top end of centrifuge container 610-   650 Starting fucan composition-   660 Arrow indicating direction of centrifugal force on container 610-   670 Centrifuge box-   900 Electrophoresis-extraction system-   910 Electrophoresis chamber-   912 Well-   914 Theoretical Displacement distances-   916 Electrophoresis gel-   918 Electrophoresis buffer-   920 Direct current power supply-   922 Cathode-   924 Anode-   926 Migration direction arrow (depicting displacement direction of    anions)-   800 Membrane dialysis system for obtaining a high-molecular-weight    fucan from a starting fucan composition-   801 Input supply line-   802 Pre-filter-   810 Fucan container-   812 Dialysis system supply line-   814 Dialysis system pump-   815 Dialyzed fluid output valve-   816 Dialyzed fluid return line-   818 Dialyzed fluid output line-   820 Dialysis cell-   825 Dialysis membrane-   830 Dialysis container-   832 Dialysate supply line-   834 Dialysate pump-   835 Dialysate fluid output valve-   836 Dialysate fluid return line-   838 Dialysate fluid output line-   840 Dialysate container-   842 Dialysate supply line-   845 Dialysate supply valve-   170 Tangential flow filtration (TFF) subsystem-   171 TFF filter-   172 TFF subsystem filter supply line-   173 TFF subsystem solvent supply valve-   174 TFF subsystem pump-   175 TFF subsystem solvent supply line-   176 TFF subsystem fucan container-   177 TFF subsystem solvent container-   178 TFF subsystem retentate line-   179 TFF subsystem permeate output line-   180 Ion exchange subsystem-   181 Ion exchange container-   182 a Ion exchange subsystem fucan supply line-   182 b Ion exchange subsystem salt solution supply line-   183 a Ion exchange subsystem fucan return valve-   183 b Ion exchange subsystem salt solution supply valve-   183 c Ion exchange subsystem salt solution return valve-   184 a Ion exchange subsystem fucan pump-   184 b Ion exchange subsystem salt solution pump-   186 Ion exchange subsystem fucan container-   187 Ion exchange subsystem salt solution container-   188 a Ion exchange subsystem fucan return line-   188 b Ion exchange subsystem salt solution return line-   189 Macroporous ion exchange resin-   300 Ion adsorption system-   301 Input supply line-   302 Inter-subsystem valve-   303 TFF subsystem retentate output line-   304 Ion exchange subsystem output valve-   305 Ion exchange subsystem output line-   306 Pre-filter

All terms used herein are used in accordance with their ordinarymeanings unless the context or definition clearly indicates otherwise.Also unless expressly indicated otherwise, in this disclosure the use of“or” includes “and” and vice-versa. Non-limiting terms are not to beconstrued as limiting unless expressly stated, or the context clearlyindicates, otherwise (for example, “including,” “having,” and“comprising” typically indicate “including without limitation”).Singular forms, including in the claims, such as “a,” “an,” and “the”include the plural reference unless expressly stated, or the contextclearly indicates otherwise.

Unless otherwise indicated, adjectives herein such as “substantially”and “about” that modify a condition or relationship characteristic of afeature or features of an embodiment, indicate that the condition orcharacteristic is defined to within tolerances that are acceptable foroperation of the embodiment for an application for which it is intended.

The scope of the present methods, compositions, systems, etc., includesboth means plus function and step plus function concepts. However, theclaims are not to be interpreted as indicating a “means plus function”relationship unless the word “means” is specifically recited in a claim,and are to be interpreted as indicating a “means plus function”relationship where the word “means” is specifically recited in a claim.Similarly, the claims are not to be interpreted as indicating a “stepplus function” relationship unless the word “step” is specificallyrecited in a claim, and are to be interpreted as indicating a “step plusfunction” relationship where the word “step” is specifically recited ina claim.

From the foregoing, it will be appreciated that, although specificembodiments have been discussed herein for purposes of illustration,various modifications may be made without deviating from the spirit andscope of the discussion herein. Accordingly, the systems and methods,etc., include such modifications as well as all permutations andcombinations of the subject matter set forth herein and are not limitedexcept as by the appended claims or other claim having adequate supportin the discussion and figures herein.

What is claimed is:
 1. A high-molecular-weight fucan wherein thehigh-molecular-weight fucan consists essentially of a molecular weightdistribution wherein at least 60% w/w of the molecular weightdistribution of the fucan is greater than about 1600 kDa when measuredusing an aqueous gel permeation chromatography set up consistingessentially of: one 300 mm analytical gel permeation chromatographycolumn with a 7.8 mm inner diameter packed with hydroxylatedpolymethacrylate-based gel, having an effective molecular weight rangeof between about 50 kDa and about 5,000 kDa, one 300 mm analytical gelpermeation chromatography column with a 7.8 mm inner diameter packedwith hydroxylated polymethacrylate-based gel, having an effectivemolecular weight range of between about 1 kDa and about 6,000 kDa andone 40 mm guard column with a 6 mm inner diameter packed withhydroxylated polymethacrylate-based gel, the two analytical gelpermeation chromatography columns and the one guard column contained ina column compartment at about 30° C.; a refractive index detector atabout 30° C.; 0.1M sodium nitrate mobile phase run at 0.6 mL/min; andquantification against a peak molecular weight standard curve consistingessentially of a first dextran standard with a peak molecular weight ofabout 2,200 kDa, a second dextran standard with a peak molecular weightof between about 720 kDa and about 760 kDa, a third dextran standardwith a peak molecular weight between about 470 kDa and about 510 kDa, afourth dextran standard with a peak molecular weight between about 370kDa and about 410 kDa, a fifth dextran standard with a peak molecularweight between about 180 kDa and about 220 kDa, and a sixth dextranstandard with a peak molecular weight between about 40 kDa and 55 kDa.2. The high-molecular-weight fucan of claim 1, wherein the sulfatecontent is between about 20% w/w and 60% w/w.
 3. Thehigh-molecular-weight fucan of claim 1, wherein the total carbohydratecontent is between about 27% w/w and 80% w/w.
 4. Thehigh-molecular-weight fucan of claim 3, wherein the total fucose contentas a percentage of the total carbohydrate content is at least about 30%w/w.
 5. The high-molecular-weight fucan of claim 3, wherein the totalgalactose content as a percentage of the total carbohydrate content isbelow about 60% w/w.
 6. The high-molecular-weight fucan of claim 3,wherein the total of glucuronic acid, mannose, rhamnose, glucose andxylose content as a percentage of the total carbohydrate content isbelow about 30% w/w.
 7. The high-molecular-weight fucan of claim 1wherein the high-molecular-weight fucan when dissolved in water at aconcentration of 50 mg/mL has a viscosity of between about 4 cP and 50cP.
 8. The high-molecular-weight fucan of claim 1 wherein thehigh-molecular-weight fucan is a white solid.
 9. Thehigh-molecular-weight fucan of claim 1 wherein the high-molecular-weightfucan when dissolved in water at a concentration from 1 mg/mL through100 mg/mL forms a solution that is clear and colorless.
 10. Thehigh-molecular-weight fucan of claim 1 wherein the fucan comprises lessthan 5% w/w acetyl content.
 11. The high-molecular-weight fucan of claim1 wherein the fucan comprises an acetyl content of substantially 0% w/wwhen measured by 2D 1H-13C heteronuclear multiple quantum coherence at70° C. with solvent signal suppression on a 600 MHz spectrometerequipped with 5-mm cold probe, in the range from 10-30 ppm in the carbondimension, in 8 increments of 256-512 scans each.
 12. A medicallyacceptable fucan composition comprising a therapeutically effectiveamount of the high-molecular-weight fucan of claim 1 in a medicallyacceptable buffer or diluent.
 13. A medical composition comprisingbetween about 0.02 mg/mL and 100 mg/mL of the high-molecular-weightfucan of claim 1, wherein the medical composition is configured andcomposed to treat a disease or condition in an animal.
 14. The medicalcomposition of claim 13 wherein the disease or condition is a fibrousadhesion.
 15. The high-molecular-weight fucan of claim 2, wherein thetotal carbohydrate content is between about 27% w/w and 80% w/w.
 16. Thehigh-molecular-weight fucan of claim 2 wherein the fucan comprises lessthan 5% w/w acetyl content.
 17. The high-molecular-weight fucan of claim3 wherein the fucan comprises less than 5% w/w acetyl content.
 18. Thehigh-molecular-weight fucan of claim 15 wherein the fucan comprises lessthan 5% w/w acetyl content.